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Fas Antigen-Mediated Apoptosis of Ovarian Surface Epithelial Cells¹

Department of Animal Science, Cornell University, Ithaca, New York 14853

Address all correspondence and requests for reprints to: Dr. Susan M. Quirk, Department of Animal Science, 258 Morrison Hall, Cornell University, Ithaca, New York 14853. E-mail: SMQ1{at}cornell.edu

	Abstract

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The Fas antigen is a cell surface receptor that, when engaged by Fas ligand or specific agonistic antibodies, triggers apoptosis. The effect of an agonistic monoclonal antibody to mouse Fas antigen (Fas mAb, clone J02) on the viability of cells from dispersed mouse corpora lutea (CL cultures) was tested. Cultures were prepared by enzymatic digestion of CL from day 4–7 pseudopregnant mice. Cultures were pretreated with 0, 1, 10, 100, or 1000 U/ml murine interferon- (IFN) at 72 h of culture. IFN has been shown to increase Fas antigen expression in a number of cell types. At 96 h (time zero), cultures were treated with Fas mAb or IgG. By 4 h after Fas mAb treatment, discrete homogeneous patches of cells within the cultures showed characteristic signs of apoptosis, including blebbing of cell membranes, detachment, and disappearance from the culture. CL cultures contain luteal, stromal, and endothelial cells; fibroblasts; and surface epithelial cells (OSE). Cells dying in response to Fas mAb were identified as OSE. Affected cells had the cobblestone appearance and distinct nuclei typical of epithelial cells. Unlike luteal cells, OSE did not stain with the lipophilic dye, Nile red. The cells did not stain with acetylated low density lipoprotein conjugated to the fluorescent marker octadecyl indocarbocyanine, a marker for endothelial cells and monocytes. Cells in patches stained positively for cytokeratin, a marker for epithelial cells. Fas-mediated cytotoxicity was quantified by counting the number of cells present in discrete patches of OSE 0 and 8 h after Fas mAb treatment. Fas mAb treatment had no effect in cultures pretreated with 0 or 1 U/ml IFN, but induced significant death of OSE in cultures pretreated with 10, 100, and 1000 U/ml IFN (37 ± 11%, 54 ± 18%, and 60 ± 11%, respectively). There was no apparent effect of Fas mAb on other cell types within the CL cultures. To confirm that cells dying in response to Fas mAb were OSE, experiments were also performed on enriched cultures of OSE prepared by enzymatic digestion of the outer surface of the ovary. In enriched OSE cultures pretreated with 200 U/ml IFN, there was 44% killing in response to Fas mAb, whereas in cells not pretreated with IFN, there was no effect. In situ fluorescent end labeling of DNA in CL cultures indicated that treatment with IFN and Fas mAb induced DNA fragmentation in OSE typical of apoptosis. Immunocytochemistry of CL cultures indicated that Fas antigen was expressed in OSE pretreated with IFN. Quantitative reverse transcriptase-PCR showed that IFN pretreatment increased Fas antigen messenger RNA levels 2.3-fold in enriched cultures of OSE. In summary, OSE in CL cultures and enriched cultures of OSE undergo apoptosis in response to Fas mAb when pretreated with IFN. In vivo, OSE undergo programmed cell death before ovulation and rapidly proliferate to repair the surface of the ovulatory follicle after ovulation. Most ovarian cancers are derived from the OSE. The results have implications for both normal ovarian function and oncogenesis in the ovary.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE OVARY is covered with a single layer of epithelial cells, the ovarian surface epithelial cells (OSE). OSE on the preovulatory follicle undergo apoptosis at the time of ovulation (1, 2) and then proliferate rapidly to repair the ruptured follicle and cover the surface of the developing corpus luteum (CL) (3). OSE are important in the oncogenesis of ovarian cancer; more than 90% of ovarian cancers arise from the OSE (4). It has been postulated that the oncogenic potential of OSE is linked to the extensive proliferation of these cells that is required during each ovulatory cycle (5). Factors affecting the death and proliferation of OSE are poorly understood. Defining these factors could provide insight into the processes by which OSE are transformed into cancerous cells.

The Fas antigen (CD95, APO-1) is a member of the tumor necrosis factor (TNF)/nerve growth factor family of cell surface receptors (6). Engagement of the Fas antigen with its ligand (Fas ligand) induces apoptosis. Northern analysis showed that Fas antigen is expressed at the highest levels in the thymus and at lower levels in the liver, heart, and ovary of the mouse (7). The Fas ligand is homologous to members of the TNF family. It is expressed at high levels on activated T lymphocytes (8) and mediates apoptosis of target cells and regulation of the immune response (6). Fas ligand is also expressed in the testis and anterior chamber of the eye, where it establishes the immune-privileged status of these tissues (9, 10), and in a number of other tissues (8, 11, 12, 13) including the ovary (12, 13). The cytotoxic function of the Fas antigen has been studied by engaging the Fas antigen with specific antibodies (Fas mAb) that mimic the effect of the natural ligand to induce apoptosis (14, 15).

Previous studies in our laboratory showed that antihuman Fas mAb induced apoptosis in human granulosa/luteal cells that were pretreated with interferon- (IFN) (16). The current study tested the effects of antimouse Fas mAb on viability of cultured mouse CL cells. CL cultures contain a number of cell types, including luteal, stromal, and endothelial cells and fibroblasts. We found that CL cultures also contain OSE; these cells proliferated in culture and formed discrete patches that were readily distinguished from surrounding cells. The data presented show that OSE express Fas antigen messenger RNA (mRNA) and protein and that Fas mAb induces apoptosis of OSE present in CL cultures and in enriched cultures of OSE. Pretreatment with IFN was required for Fas-mediated cytotoxicity and was associated with increased expression of Fas antigen by OSE. Other cell types present in the CL cultures appeared to be unaffected. This study identifies the OSE as a Fas antigen-sensitive cell type in the mouse ovary.

	Materials and Methods

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Materials
CD1 mice were obtained from Charles River (Wilmington, MA). Culture medium, FBS, penicillin, streptomycin, fungizone, transferrin, insulin, and sodium selenite were obtained from Life Technologies (Grand Island, NY). BSA, deoxyribonuclease I, sodium pyruvate, L-glutamine, Nile red, and Triton X-100 were purchased from Sigma Chemical Co (St. Louis, MO). Tissue culture plates were obtained from Corning-Costar (Cambridge, MA), except for Slide-well chambers, which were obtained from Nunc-Intermed (Naperville, IL). Monoclonal hamster antimouse Fas antigen antibody (clone Jo-2) was a gift from S. Nagata, Osaka University Medical School (Osaka, Japan). Murine IFN was purchased from Genzyme (Cambridge, MA). Octadecyl indocarbocyanine (DiI)-acetylated low density lipoprotein (acLDL) and avidin-BODIPY FL conjugate were obtained from Molecular Probes (Eugene, OR). Rabbit antimouse Fas antigen antibody for immunocytochemistry was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and rabbit antihuman cytokeratin was obtained from Biodesign International (Portland, ME). Biotinylated goat antirabbit IgG and avidin-fluorescein isothiocyanate conjugate were obtained from Jackson Immunochemicals (West Grove, PA). Murine epidermal growth factor, terminal deoxynucleotidyl transferase, and avian myeloblastosis virus reverse transcriptase (RT) were obtained from Promega (Madison, WI), random hexamer was obtained from Pharmacia (Piscataway, NJ), Taq polymerase was obtained from Fisher (Pittsburgh, PA), and biotin deoxy-UTP (dUTP) and collagenase/dispase were obtained from Boehringer Mannheim (Indianapolis, IN).

Culture and animals
CL cultures.
Ovaries were obtained from pseudopregnant CD1 mice 4–6 days postbreeding with a vasectomized male. Procedures were approved by the Cornell University Institutional Animal Care and Use Committee and are in accord with the NIH Guide for the Care and Use of Laboratory Animals. Ovaries were dissected, placed in DMEM-Ham’s F-12 medium (DMEM-F12), and trimmed. Individual CL were isolated under a dissecting scope by gently teasing them free from the surrounding tissue. Isolated CL were digested with collagenase/dispase (4 mg/ml) containing 10 µg/ml deoxyribonuclease I and 10 mg/ml BSA for 90 min at 37 C. The cell suspension was gently triturated every 30 min using pulled Pasteur pipettes with successively smaller bore sizes. The resulting cells were primarily in clumps of 2–10 cells. Cells were resuspended in basal medium (DMEM-F12 containing 10% FBS, 88 µg/ml pyruvate, 292 µg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone) and plated at a density of 4 x 10⁴ cells/well in 24-well culture plates. For cytochemical analyses, cells were plated at the same concentration in 8 x 20-mm Slide-well chambers. Media were changed at 2-day intervals.

Enriched OSE cultures.
Whole ovaries were isolated from pseudopregnant mice as described above. The surface of the ovary was digested in collagenase/dispase (described above) for 45–60 min at 37 C, followed by gentle vortexing for 2–4 min. Ovaries were discarded, and the suspended cells were washed twice and resuspended in OSE growth medium (DMEM-F12 containing 5% FBS supplemented with 5 µg/ml transferrin, 2 µg/ml insulin, 3.5 ng/ml sodium selenite, 0.5 µg/ml hydrocortisone, and 10 ng/ml epidermal growth factor). Before culture, plastic culture dishes were coated with gelatin (calf gelatin; bloom, >250) by incubating a 1% solution in each well for 60 min at 37 C and rinsing with DMEM-F12. OSE were plated at a density of 2 x 10⁴ cells/well in 24-well dishes or in Slide-well chambers. Media were changed at 2-day intervals.

Fas mAb-induced cell death
The responsiveness of CL cultures to Fas-mediated death was assessed by incubation with a hamster antimurine Fas mAb (clone Jo-2) that triggers cell death when bound to the Fas antigen (17). CL cultures were preincubated with 0, 1, 10, 100, or 1000 U/ml IFN at 72 h of culture. At 96 h (time zero), Fas mAb or hamster IgG was added at a concentration of 1 µg/ml. In preliminary experiments, only small differences in viability, as assessed by 3-(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium bromide (MTT) (16) assay, were noted, but significant cell death was visibly observed in patches of OSE. To determine the percentage of cell death occurring in OSE, individual cells in distinct patches of OSE were counted at 0 h and again at 8 h. Each treatment was performed in three wells, and two patches were counted in each well (mean no. of cells/patch, 62). The experiment was replicated three times using separate CL preparations. Killing of OSE by Fas mAb in enriched cultures of OSE was quantified similarly, except that IFN was added to cultures at concentrations of 0 and 200 U/ml on day 3 or 4 of culture. At this time OSE were found in small patches and represented 50–80% of the cells. The remaining cells were predominantly stromal cells. Each treatment was analyzed in two patches from each of three wells, and the experiment was repeated using three separate culture preparations.

Cell counts were analyzed by a mixed model, repeated measures ANOVA with treatment as the random variable and experiment as the fixed variable (18).

Cytochemistry
CL cultures were incubated with DiI-acLDL, which is taken up specifically by endothelial cells and monocytes (19). Cells were incubated with DiI-acLDL (10 µg/ml in DMEM-F12) for 4 h, rinsed with DMEM-F12, and examined for epifluorescence using a Nikon Diaphot 200 microscope with a 546 nm excitation filter and a 590 nm absorption filter (Nikon, Tokyo, Japan).

CL cultures were stained for lipid by use of Nile red (20). Nile red (500 µg/ml in acetone) was diluted 100-fold in 0.01 M PBS, added to cells for 5 min, and rinsed with PBS. Epifluorescence was viewed using a 546-nm excitation filter and a 590-nm absorption filter.

CL cultures were immunostained for cytokeratin and Fas antigen. Cells were fixed for 15 min at -20 C in Carnoy’s fixative, blocked by incubation with PBS containing 0.3% Triton X-100 and 2% normal goat serum (NGS) for 30 min at 25 C, and incubated with rabbit polyclonal antihuman cytokeratin, which binds to cytokeratins from a variety of species, including the mouse, or with rabbit polyclonal antimouse Fas antigen antibody. Nonspecific binding was assessed with normal rabbit serum and rabbit IgG, respectively. Blocking buffer was used as diluent. After washing, cells were incubated with biotinylated goat antirabbit IgG followed by an avidin-fluorescein isothiocyanate conjugate. Epifluorescence was viewed using a 495-nm excitation filter and a 520-nm absorption filter.

In situ end labeling of DNA
In situ end labeling of cellular DNA was used to detect fragmentation of DNA typical of apoptosis (21). CL cultures in slide wells were pretreated with IFN for 24 h, followed by addition of Fas mAb or hamster IgG as described above. Eight hours after treatment, cells were fixed in Carnoy’s fixative for 15 min at -20 C and hydrated in PBS. Cells were incubated with 10 µM biotin-dUTP and 200 U/ml terminal deoxynucleotidyl transferase enzyme for 30 min at room temperature, rinsed, blocked with PBS-1% NGS for 5 min, incubated with avidin-BODIPY FL in PBS-1% NGS, and observed under phase contrast and epifluorescent illumination using a 495-nm excitation filter and a 520-nm absorption filter.

Analysis of Fas antigen mRNA
Fas antigen mRNA was quantified by a competitive RT-PCR assay. Enriched cultures of OSE at 7–11 days of culture were incubated with 0 or 200 U/ml IFN for 24 h, and total RNA was isolated (22). RNA was prepared from three independent sets of enriched OSE cultures that were generated using different pools of mice. RNA (1 µg) was reverse transcribed in the presence of various concentrations of an internal standard RNA (0.19–23.26 attomoles/reaction) using avian myeloblastosis virus RT and random hexamer primer. The internal standard RNA was prepared by in vitro transcription of a 634-bp fragment of mutated Fas antigen complementary DNA (cDNA) in the plasmid pALTER-1 (Promega) containing a 50-bp deletion internal to the PCR primer-binding sites (positions 500–549; numbering according to Ref.7). A fifth of the cDNA in the RT reaction was amplified by PCR in the presence of [³²P]dCTP. Amplification consisted of a preincubation at 94 C for 1 min before adding Taq polymerase and then 40 cycles at 94 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec. Primers were designed to generate a 264-bp fragment for the test RNA and a 214-bp fragment for the internal RNA standard (the positions of 5'- and 3'-primers were from 368–397 and from 631–602, respectively). RT-PCR products were fractionated on a 2% agarose gel. The gel was dried, and radioactive signal was quantified on a Fuji BAS1000 phosphoimager (Fuji, Tokyo, Japan). The concentration of Fas antigen mRNA in each OSE sample was calculated by regression of the log signal ratio sample:standard vs. the standard concentration. The sample concentration equals the standard concentration at the point where the sample signal equals the standard signal. The sample concentration is corrected for the 50-bp difference in the length of PCR products between the sample and the standard. Samples from the same culture preparation were assayed together. The slopes of signal ratio vs. standard concentration for samples from the same culture preparation were tested and found to be parallel based on overlap of 95% confidence intervals of the calculated slopes. The calculated sample concentrations were within the range of standard concentrations and were not extrapolated. The between-assay coefficient of variation was 11.0 ± 3.5%. Data were analyzed by paired t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of OSE in cultures of dispersed mouse CL
Twenty-four hours after plating dispersed CL cells, cultures consisted primarily of luteal cells and stromal cells, with lesser numbers of fibroblasts and endothelial cells. Occasional small patches of epithelial-type cells (usually <10 cells/patch) were observed. These cells were cuboidal in shape, had rounded nuclei containing 2–3 nuclear bodies, and were found in distinct, usually round, patches devoid of other cell types. By 72 h, these patches were clearly visible (5–15 patches/well), usually containing 20–200 cells/patch (Fig. 1). Positive identification of the cells in patches as derived from the ovarian surface epithelium was based on the following criteria. 1) The cells had the cobblestone appearance and distinct nuclei typical of epithelial cells (Fig. 1). 2) Patches of cells identical to those observed in CL cultures were obtained by digesting the outer cell layers of the ovary, where OSE are found (Fig. 1). OSE in enriched cultures grew in patches similar to those seen in CL cultures and formed large confluent areas after 1–2 weeks. 3) Luteal cells and stromal cells stained positively for the lipophilic fluorescent dye, Nile red, indicative of steroid-containing granules, whereas the OSE did not (Fig. 2). 4) Cells in patches did not stain with DiI-acLDL, which is taken up specifically by endothelial cells and monocytes (19). Sporadic cells outside the OSE patches, presumably endothelial cells, fluoresced brightly. Control cultures of bovine aortic endothelial cells showed bright staining (data not shown). 5) Cells in patches stained positively for cytokeratin, a marker for epithelial cells (5), whereas other cell types did not (Fig. 2).

View larger version (177K): [in this window] [in a new window]   Figure 1. Morphological appearance of OSE. A culture prepared by enzymatic dispersion of mouse CL (CL culture), 72 h after plating, is shown in a. A well defined, round patch of OSE is present in the center of a (magnification, x200). A culture prepared by enzymatic digestion of the outer cell layer of the ovary (enriched OSE culture), 11 days after plating, is shown in b (magnification, x400).

View larger version (135K): [in this window] [in a new window]   Figure 2. Identification of patches of OSE in CL cultures. CL cultures were stained with the lipophilic dye, Nile red (a and b; magnification, x200), or with antibody against cytokeratin followed by antirabbit IgG-FITC secondary antibody (c and d; magnification, x400). The top panels show phase contrast microscopy, and the bottom panels show fluorescence microscopy. Experiments were repeated at least three times.

View larger version (21K): [in this window] [in a new window]   Figure 3. Fas mAb-induced death of OSE in CL cultures. CL cultures were preincubated with 0, 1, 10, 100, or 1000 U/ml IFN at 72 h of culture. At 96 h (time zero), Fas mAb or hamster IgG (1 µg/ml) was added. Cells in individual patches of OSE were counted at 0 and 8 h, and the percentage of killing was calculated. Bars represent the mean ± SEM of three experiments using separate CL preparations. In each experiment treatments were repeated in three wells, and two patches were counted per well. *, P < 0.05; **, P < 0.01 (vs. IgG control).

View larger version (178K): [in this window] [in a new window]   Figure 4. Morphological features of Fas antigen-mediated death of OSE in CL cultures. CL cultures were pretreated with 100 U/ml IFN (a and b) or 1000 U/ml IFN (c and d) at 72 h of culture. a, c, and e show OSE patches at 96 h of culture (time zero). b, d, and f show the same OSE patches 8 h after treatment with 1 µg/ml Fas mAb (b and d) or hamster IgG (f). Magnification, x200.

View larger version (17K): [in this window] [in a new window]   Figure 5. Fas mAb-induced death of enriched cultures of OSE. Three or 4 days after plating, cells were pretreated with 0 or 200 U/ml IFN for 24 h. Fas mAb or hamster IgG was then added (time zero). The number of cells in confluent patches of OSE was counted at 0 and 8 h, and the percentage of killing was calculated. Bars represent the means ± SEM of three experiments using separate OSE preparations. In each experiment treatments were repeated in three wells, and two patches were counted per well. * P < 0.01 vs. IgG control.

View larger version (123K): [in this window] [in a new window]   Figure 6. Immunocytochemistry of Fas antigen expression by OSE in CL cultures. Cultures were treated with IFN at 72 h of culture. After 24 h, cultures were fixed and incubated with a polyclonal rabbit antimouse Fas antigen antibody (a and b) or IgG (c and d). Binding was detected by antirabbit IgG-FITC secondary antibody. The top panels show phase contrast microscopy, and the bottom panels show fluorescence microscopy. Magnification, x400. This procedure was repeated four times.

View larger version (32K): [in this window] [in a new window]   Figure 7. Quantitative competitive RT-PCR assay of Fas antigen mRNA expression by enriched cultures of OSE treated with and without IFN for 24 h. RNA was prepared from OSE cultures and reverse transcribed in the presence of increasing amounts of an internal Fas antigen RNA standard. The resulting cDNA was amplified using primers that generated a 264-bp fragment from the wild-type (WT) Fas antigen mRNA and a 214-bp fragment from the RNA standard. A, Phosphoimage of the 32P-labeled PCR products in a representative experiment. B, Logarithmic plot of the data shown in A. Complete standard curves were generated for each RNA sample (four points, with one replicate per point), and the regression line was calculated to determine the concentration of Fas antigen mRNA. C, Summary of Fas antigen mRNA levels in control and IFN-treated OSE cultures (n = 3 independent sets of enriched OSE cultures prepared from different mice; mean ± SEM). *, P < 0.005.

View larger version (109K): [in this window] [in a new window]   Figure 8. Detection of DNA fragmentation by in situ end labeling of DNA. CL cultures were pretreated with IFN at 72 h of culture and then treated with Fas mAb (a and b) or hamster IgG (c and d) at 96 h (time zero). At 8 h, cells were fixed, and DNA fragmentation was detected by end labeling DNA as described in Materials and Methods. The top panels show phase contrast microscopy, and the bottom panels show fluorescence microscopy. Magnification, x400. This procedure was repeated three times.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IFN increased levels of Fas antigen mRNA and expression of immunoreactive Fas antigen protein in OSE, and pretreatment with IFN was necessary for Fas mAb-induced apoptosis. Binding of Fas ligand to Fas antigen is thought to induce trimerization of the receptor and subsequent signaling of apoptosis (6). Levels of Fas antigen expression in OSE not pretreated with IFN may be insufficient to allow trimerization of the Fas antigen. However, IFN could also be affecting other aspects of the cell death pathway, as significant Fas antigen mRNA was present in non-IFN-treated cells, and IFN induced a modest (2.3-fold) increase in Fas antigen mRNA. Previous studies showed that antihuman Fas mAb induced apoptosis of human granulosa/luteal cells only when cells were pretreated with IFN (16). IFN increased the expression of Fas antigen in various cell lines (7, 14, 25, 26, 27, 28) and in primary cultures of rat granulosa cells (13), thyroid cells (29), and keratinocytes (30), and enhanced Fas antigen-induced apoptosis. It is not known whether IFN is a physiological trigger for Fas antigen expression in the ovary or whether another factor(s) might regulate Fas antigen expression and/or responsiveness. However, IFN has been shown to have other effects on ovarian function, including inhibition of LH receptor induction (31), gonadotropin-stimulated steroidogenesis (31, 32, 33, 34), and inhibin production (34) by granulosa cells and inhibition of progesterone secretion by bovine luteal cells (35). Infiltration by leukocytes occurs as part of the normal physiology of the ovary (36), and these cells could provide a source of IFN; T lymphocytes and IFN were found in the follicular fluid of human ovaries (37, 38). Interleukin-1ß and TNF have also been shown to increase Fas antigen expression in a number of cell types (25, 26, 28, 29), and these cytokines are produced by ovarian cells (39, 40). Physiologically relevant regulators of Fas antigen expression in the ovary remain to be determined.

OSE undergo cycles of proliferation and degeneration associated with ovulation. Before ovulation, OSE in apposition to the follicular apex degenerate and are removed by the process of apoptosis (1, 2, 41). It is not known whether the Fas antigen plays a physiological role in this degeneration. However, the current results indicate that the pathway for Fas-induced death is functional in OSE, at least when stimulated by IFN.

After ovulation, OSE proliferate rapidly to cover the surface of the newly formed CL (3). The proliferative activity of the OSE has been suggested to be linked to the high frequency of ovarian cancer arising from the OSE. Continual cycles of proliferation are thought to provide increased chances for mutagenic changes resulting in cancer (4, 5). A number of human ovarian cancer cell lines have been tested for their susceptibility to Fas-induced death. In cell lines classified as either sensitive or resistant to Fas-induced death, IFN was generally effective in increasing the susceptibility to Fas mAb. However, some cell lines remained resistant to Fas mAb in the presence of IFN (27). The possibility that resistance to Fas-induced death contributes to ovarian cancer is worthy of further study.

A necessary factor for Fas-induced apoptosis in the ovary is a source of Fas ligand. Fas ligand is expressed most abundantly on activated T cells. It is one of the major effectors used by cytotoxic T cells to kill target cells and is required for lymphocyte selection and regulation of the immune response (6). Fas ligand is a transmembrane protein that can interact with the Fas antigen in the membrane-bound form or in a soluble form, consisting of the extracellular portion of the Fas ligand that has been cleaved from the membrane (42). Fas ligand mRNA was detected by Northern analysis at relatively high levels in rat splenocytes, thymocytes, and testis and at moderate levels in small intestine and lung (8). Fas ligand mRNA was detected in the ovary at low levels using ribonuclease protection analysis (12). Leukocytes that infiltrate the ovary (36) could potentially provide a source of Fas ligand. In addition, Fas ligand may be expressed by ovarian cells. One study demonstrated immunoreactive Fas ligand expression by rat oocytes, but found no evidence for Fas ligand expression in other ovarian cell types (13).

Luteal cells isolated between days 4 and 6 of pseudopregnancy are unresponsive to Fas mAb. Immunocytochemistry showed that IFN increased Fas antigen expression in patches of OSE present in CL cultures, but not in other cells. Therefore, it is possible that CL cells at this stage of pseudopregnancy do not express Fas antigen at a level sufficient to respond to Fas mAb. We cannot rule out the possibility that CL cells express Fas antigen at levels below the sensitivity of immunocytochemistry. Along these lines, RT-PCR detected Fas antigen mRNA in enriched cultures of OSE that were not pretreated with IFN despite the fact that immunocytochemistry failed to unequivocally detect staining for Fas antigen in untreated OSE. However, CL cells clearly do not express Fas antigen at levels comparable to OSE in response to IFN.

Previous studies in our laboratory showed that antihuman Fas mAb induced apoptosis of human granulosa/luteal cells pretreated with IFN. Additional pretreatment with CG increased the cytotoxic response to Fas mAb 170% over that obtained with IFN pretreatment alone (16). It was postulated that hCG-induced luteinization of the cells in culture may have enhanced Fas-induced apoptosis. A recent report demonstrated that Fas antigen expression increases during luteal development in humans (43). Failure of IFN-pretreated mouse CL cells to die in response to Fas mAb in the present study may represent a species difference or may be due to differences in the differentiation of the cells. A variety of cell types have been reported to be resistant to Fas-mediated death despite expression of Fas antigen (44, 45, 46, 47, 48). Resistance vs. sensitivity to Fas-mediated killing appears to be dependent upon the stage of differentiation or activation of the cell. For example, lymphocytes that express Fas antigen must be activated by treatment with interleukin-2 to acquire responsiveness to Fas antigen-induced apoptosis (49). Complex intracellular pathways, involving a number of interacting cytoplasmic death domain proteins that are downstream effectors of Fas-mediated death (50) as well as proteins that inhibit Fas-induced death (51), have been identified. The balance of these factors within the cell may determine the sensitivity to Fas-mediated killing.

In summary, OSE undergo apoptosis in response to engagement of the Fas antigen when pretreated with IFN. A role for the Fas antigen in regulating physiological turnover of the OSE and its potential involvement in the etiology of ovarian cancer are areas for future study.

	Acknowledgments

 
The authors thank Shigekazu Nagata, Osaka University Medical School, for providing the antimouse Fas antigen antibody clone Jo2.

	Footnotes

 
¹ This work was supported by NIH Grant HD-32535–02.

Received April 7, 1997.

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