| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
, Tumor Necrosis Factor-
, and Cycloheximide1
Department of Animal Science, Cornell University, Ithaca, New York 14853
Address all correspondence and requests for reprints to: Dr. Susan M. Quirk, 258 Morrison Hall, Cornell University, Ithaca, New York 14853. E-mail: smq1{at}cornell.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(TNF) receptor family that, when activated by Fas
ligand or agonistic antibodies, induces death by apoptosis. Although
the presence of Fas antigen in ovarian tissues has been demonstrated,
little is known about whether Fas antigen is functional in the ovary.
This report shows that murine granulosa cells are initially resistant
to antibody-induced Fas-mediated apoptosis, but will undergo apoptosis
when cotreated with TNF and interferon-
(IFN) or cycloheximide (CX).
Granulosa cells were obtained from follicles of 23-day-old mice 2 days
after injection of PMSG. Twenty-four hours after plating, cells were
pretreated with either 0 or 200 U/ml IFN, which has been shown to
induce Fas antigen expression and is required for Fas-mediated killing
in many cell types. At 48 h, cells were treated with 2 µg/ml
control IgG, 2 µg/ml anti-Fas antigen antibody (Fas mAb), 10 ng/ml
TNF, or Fas mAb and TNF. Cytotoxicity (percent killing) relative to
control IgG was determined at 72 h by counting granulosa cells
after trypsinization. In the absence of IFN, no cytotoxicity was
observed. In the presence of IFN, neither TNF or Fas mAb alone was
cytotoxic, but the combination of Fas mAb and TNF resulted in 25%
killing (P < 0.05). Fas antigen messenger RNA
(mRNA) was detectable in cultures not treated with cytokines and was
increased 5-fold by TNF, 2-fold by IFN, and 17-fold by the combination
of IFN and TNF. To test whether the presence of a labile inhibitor(s) of Fas-mediated killing in granulosa cells is the cause of resistance to Fas mAb, the protein synthesis inhibitor CX was used. Experiments were performed as described above, except that cells were treated with 0.5 µg/ml CX in conjunction with other treatments at 48 h. Fas mAb treatment in the presence of CX induced 25% cell death without IFN pretreatment and 38% with IFN (P < 0.05). TNF treatment in the presence of CX had no effect alone, but potentiated the effects of Fas mAb, resulting in 56% killing in the absence of IFN and 86% killing in the presence of IFN (P < 0.05). Cells stained positively for DNA fragmentation and annexin V binding, features characteristic of apoptosis.
Because initial experiments showed that treatment with TNF alone increased Fas mRNA levels, the effect of pretreating cells for 24 h with TNF before treatment with Fas mAb was tested. Pretreatment with TNF or IFN alone did not promote Fas mAb-mediated killing, but combined pretreatment with TNF and IFN resulted in 25% killing in response to Fas mAb. Treatment of cells with the combination of IFN and TNF induced a 19-fold increase in Fas antigen mRNA levels. Corresponding increases in Fas antigen protein expression on the surface of cells in response to cytokine treatments were detected by immunocytochemistry. Human TNF did not duplicate the effects of mouse TNF in inducing Fas antigen mRNA expression and Fas mAb-induced killing. As human TNF interacts exclusively with the type I, but not the type II, TNF receptor in the mouse, potentiating effects of mouse TNF on the Fas pathway are probably mediated via the type II TNF receptor.
The effects of cytokine treatments on levels of mRNA for FAP-1, an inhibitor of Fas-mediated apoptosis, were determined. FAP-1 mRNA was detectable in untreated granulosa cells, and levels were not altered by treatment with TNF and/or IFN.
In summary, the Fas-mediated pathway of apoptosis is functional in mouse granulosa cells that are stimulated with IFN and TNF. These cytokines may function at least partially by increasing Fas antigen expression. Granulosa cells appear to have inhibitors of the Fas antigen pathway, as treatment with CX potentiates Fas-mediated death. TNF promotes Fas-mediated killing in the presence and absence of CX. Therefore, TNF is not likely to act simply by increasing Fas antigen expression or decreasing protein inhibitors of the Fas pathway, because TNF remains effective when these processes are blocked by CX.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, basic fibroblast growth factor, and
interleukin-1ß. In addition, members of the bcl-2 family
have been implicated as either suppressors or initiators of ovarian
cell apoptosis (1). Factors reported to induce apoptosis of ovarian
cells include GnRH (2), tumor necrosis factor- (TNF) (3, 4),
androgen (5), and Fas ligand (FasL) (6, 7). The current study examined
whether granulosa cells from the mouse are sensitive to Fas-mediated
apoptosis and the modulation of the Fas pathway by the cytokines,
interferon- (IFN), and TNF. The Fas antigen (APO-1, CD95) is a transmembrane receptor that belongs to the TNF/nerve growth factor receptor family. Sensitive cells that express the Fas antigen undergo apoptosis upon contact with FasL or agonistic, cross-linking antibodies. FasL is a type II membrane protein that, like TNF, can be processed to a soluble form by a metalloproteinase (8). Fas antigen and FasL are expressed in the ovary (9, 10). Variable reports indicate expression of Fas antigen in one or more locations in the ovary, including the granulosa, theca, oocyte, corpus luteum, and surface epithelium (6, 7, 11, 12, 13, 14, 15). Expression of FasL has been reported in oocytes (13), granulosa cells (15, 16), and thecal cells (15). Our previous studies showed that engagement of the Fas antigen with agonistic anti-Fas antigen antibodies induced apoptosis in human granulosa/luteal cells that were pretreated with IFN (6) and in ovarian surface epithelial cells of the mouse that were pretreated with IFN (7). Thus, the pathway for Fas-mediated death may be active in some ovarian cells under certain conditions.
TNF has pleiotropic effects in many cell types, inducing differentiation, proliferation, and apoptosis (17, 18). TNF is produced by ovarian macrophages, endothelial cells, granulosa cells, thecal cells, luteal cells, and oocytes and modulates a variety of functions in the ovary (19). Inhibitory effects of TNF on ovarian cell viability as well as proliferative effects have been reported (19).
IFN and TNF have been shown to increase Fas antigen expression in a number of cell types and to increase Fas-mediated apoptosis (7, 11, 13, 20, 21). Although the pathways for Fas- and TNF-induced apoptosis are distinct, they share some common interacting components (8). The cytoplasmic death domain of the Fas antigen binds a protein, FADD/MORT, that then recruits a cysteine protease, or caspase, known as FLICE/MACH to the receptor complex. This initiates a cascade in which a series of caspases becomes activated, resulting in cleavage of cellular substrates, activation of an endonuclease, and cell death (8, 22). The cytoplasmic death domain of the type I TNF receptor (TNFRI) binds TRADD, a protein that then recruits FADD to the TNFRI complex. The Fas- and TNF-mediated pathways converge at this point. Signal transduction by FasL and TNFRI also involves the stimulation of sphingomyelinase to generate the second messenger ceremide, which induces apoptosis in many cell types (8). Recent studies have identified several inhibitors of the Fas antigen-mediated pathway for apoptosis (23, 24). One of these, FAP-1, is expressed at highest levels in cell types that are resistant to Fas-mediated apoptosis (23). The present study investigates interactions among Fas antigen, TNF, and IFN to promote granulosa cell apoptosis and the presence of protein inhibitors of the Fas pathway in granulosa cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell culture: animals
Ovaries were obtained from 22-day-old mice 2 days after the
injection of 10 IU PMSG. 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, and trimmed.
Ovaries had numerous, large preovulatory follicles and a complete
absence of corpora lutea. Large follicles were punctured with a
26-gauge needle. Granulosa cells expressed into the media were
collected by centrifugation, washed, briefly triterated, and counted.
The resulting cells were primarily in clumps of 2–10 cells. Cells were
plated on day 0 in DMEM-Ham’s F-12 medium containing 10% FBS plus
penicillin, streptomycin, and fungizone at concentrations of 8 x
104 cells/well in 24-well culture dishes for killing
assays, 8 x 104 cells/well in 4-chamber Slide-wells,
and 4 x 105 cells/well in 6-well culture dishes for
RNA analysis. Media were changed daily, and all treatments and
pretreatments were added in DMEM-Ham’s F-12 containing 5% FBS plus
antibiotics.
Cell cultures: experimental design
In the first experiment, the ability of Fas mAb and murine TNF
to induce cell death was assessed. Granulosa cell cultures were
preincubated with 0 or 200 U/ml IFN on day 1 of culture. On day 2,
cultures were given the following treatments in the presence or absence
of IFN: 1) 2 µg/ml Fas mAb; 2) 2 µg/ml hamster IgG, as a control);
3) 10 ng/ml TNF; or 4) TNF plus Fas mAb. Pretreatment and treatment
were performed with fresh medium changes. On day 3, trypsin was added,
and live cells were counted in a hemocytometer by trypan blue
exclusion. In additional cultures, cells pretreated with or without IFN
on day 1 and with or without TNF on day 2 were frozen at -80 C on day
3 for subsequent analysis of Fas antigen messenger RNA (mRNA). The
percent killing for Fas mAb- and TNF-treated cells was based on cell
counts of control (IgG) wells given the same pretreatment. All
treatments were performed in three wells, and the experiment was
replicated three times using separate granulosa cell preparations. The
percent killing was analyzed by a one-way randomized complete block
ANOVA, with Duncan’s procedure used to compare individual means (25).
To test for effects on viability due to pretreatment, cell numbers in
the control (IgG-treated) group pretreated with IFN were compared with
cell numbers in the control group not pretreated with IFN by paired
t test.
To study the presence of labile protein inhibitors of Fas-mediated cell death, experiments were performed in the presence of cycloheximide (CX), an inhibitor of protein synthesis. The experiment was performed exactly as described above except that cells were pretreated with 0.5 µg/ml CX 2 h before treatment with Fas mAb or TNF. The dose of CX was chosen based on preliminary dose-response studies as the highest concentration that did not cause significant cell death (data not shown). Separate control wells without CX were used to assess the effects of CX. The medium was changed with CX treatment, and Fas mAb and TNF were added in small volumes to the existing medium. All treatments were replicated in three wells, and the entire experiment was repeated with three separate granulosa cell preparations. Data were analyzed as described above. In addition, cell numbers in control wells not receiving CX were compared with numbers in control wells receiving CX by randomized complete block ANOVA and Duncan’s procedure (25).
A third series of experiments tested the effects of pretreatment with TNF (and/or IFN) on Fas-mediated cell death. Day 1 cultures were pretreated with medium (control), 10 ng/ml TNF, 200 U/ml IFN, or IFN plus TNF. On day 2, medium was replaced with medium containing the same pretreatment with either Fas mAb (2 µg/ml) or hamster IgG (2 µg/ml) as the control. Twenty-four hours after treatment, cells were counted as described above. All treatments were replicated in three wells, and the entire experiment was repeated with three separate granulosa cell preparations. Additional cultures were treated as described on day 1 and frozen at -80 C on day 2 for subsequent analysis of Fas antigen mRNA. For each pretreatment, the percent killing by Fas mAb was calculated, and differences in percent killing were analyzed by a randomized complete block ANOVA and Duncan’s procedure. To identify the effects of pretreatment alone on viability, cell numbers in the IgG-treated wells of each pretreatment were compared by randomized complete block ANOVA.
To test whether TNF was affecting Fas-mediated killing through the TNFRI or the type II receptor (TNFRII), the effect of human TNF (hTNF) was examined. hTNF binds to murine TNFRI, but not murine TNFRII (26). Day 1 cultures were pretreated with medium (control), 10 ng/ml hTNF, or 200 U/ml IFN plus hTNF. On day 2, medium was replaced with medium containing the same pretreatment with or without Fas mAb (2 µg/ml), and cells were counted 24 h after treatment. Experiments were repeated three times as described above. Additional cultures were treated with either medium or 10 ng/ml hTNF on day 1 and were frozen at -80 C on day 2 for analysis of the Fas antigen mRNA concentration. The percent killing was analyzed as described in the previous experiment.
Analysis of Fas antigen mRNA
Fas antigen mRNA was quantified by a competitive RT-PCR
assay as previously described (7). The assay uses an internal RNA
standard that was prepared by in vitro transcription of a
634-bp fragment of mutated Fas antigen complementary DNA (cDNA) in the
plasmid pALTER-1 containing a 50-bp deletion internal to the PCR
primer-binding sites (positions 500–549; numbering according to Ref.
9). SP6 RNA polymerase was used in a reaction containing 2 µg DNA
template according to the manufacturer’s recommendations. The reaction
was treated with phenol-chloroform and ethanol precipitated, and the
concentration of RNA was determined by spectrophotometry at OD 260.
Total RNA was prepared from cultured cells (27), and 1 µg was reverse
transcribed in the presence of various concentrations of the internal
standard RNA (2–250 attomoles/reaction) using AMV RT and random
hexamer primer. cDNA in the RT reaction was amplified by PCR in the
presence of [32P]dCTP using primers 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 in exons 3 and 4 and from 631–602 in exons 7 and 6,
respectively) (9). RT-PCR products were fractionated on a 2% agarose
gel. The gel was dried, and radioactive signal was quantified on a
Fuji Medical Systems USA, Inc. (Stamford, CT) BAS1000
phosphorimager. The concentration of Fas antigen mRNA in each sample
was calculated by regression of the log of the ratio of sample signal
intensity to standard signal intensity vs. 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 a one-way randomized complete block ANOVA with
supplemental analyses by Duncan’s procedure (25).
Analysis of FAP-1 mRNA
FAP-1 mRNA was quantified by a RT-PCR assay similar to the Fas
antigen assay. Briefly, RNA (1 µg) was reverse transcribed in the
presence of various concentrations of an internal standard RNA
(24–3000 attomoles/reaction) as described. The internal standard RNA
was prepared by in vitro transcription of a 242-bp fragment
of mutated FAP-1 cDNA in the plasmid pALTER-1 containing a 60-bp
deletion internal to the PCR primer binding sites (positions
4167–4226; numbering according to GenBank accession no. Z32740). cDNA
in the RT reaction was amplified by PCR in the presence of
[32P]dCTP using primers designed to generate a 302-bp
fragment for the test RNA and a 242-bp fragment for the internal RNA
standard (positions of 5'- and 3'-primers were from 4036–4059 and from
4337–4315, respectively; GenBank accession no. Z32740). Amplification
consisted of preincubation at 94 C for 5 min before addition of
Taq polymerase followed by 30 cycles at 94 C for 30 sec, 63
C for 30 sec, and 72 C for 30 sec. The positions of introns in the
FAP-1 gene are not known. Signals generated by RT-PCR were from RNA
rather than genomic DNA, because control reactions in which AMV-RT was
omitted had no signal. RT-PCR products were fractionated and analyzed
exactly as described for the Fas antigen mRNA. Data were analyzed by a
one-way randomized complete block ANOVA, with supplemental analysis by
Duncan’s procedure (25).
Cytochemistry
Expression of Fas antigen protein in granulosa cell
cultures was detected by immunohistochemistry as previously described
(7). Briefly, cells were fixed for 15 min at -20 C in Carnoy’s
fixative. After blocking with PBS containing 0.3% Triton X-100 and 2%
normal goat serum (NGS), cells were incubated with rabbit polyclonal
antimouse Fas antigen antibody or rabbit IgG. Blocking buffer was used
as diluent. After washing, cells were incubated with biotinylated goat
antirabbit IgG followed by Neutravidin-Bodipy FL.
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 (7, 28). 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 TdT enzyme
for 30 min at room temperature, then rinsed, blocked with PBS-1% NGS
for 5 min, incubated with Neutravidin-Oregon Green 495 in PBS-1% NGS,
and observed under phase contrast and epifluorescent illumination using
a 495-nm excitation filter and a 520-nm absorption filter.
Detection of membrane-associated phosphatidylserine
Detection of phosphatidylserine on the outside of the cell
membrane, a unique and early marker for apoptosis (29), was performed
using a commercial kit (TACS Annexin V-Oregon Green, Trevigen,
Gaithersburg, MD). Cells were cultured as described above and were
tested 5 h after Fas mAb treatment in the presence of CX or 9
h after Fas mAb treatment in the absence of CX. Binding of annexin
V-Oregon Green conjugate (dilution = 1:50) and propidium iodide
(PI; dilution = 1:20) were performed according to the
manufacturer’s instructions. After binding and washing, cells were
fixed in methanol at -20 C for 20 min, hydrated for 5 min in PBS, and
coverslipped. Cells were observed under phase contrast and
epifluorescent illumination using a 495-nm excitation filter and a
520-nm absorption filter for annexin V-Oregon Green and a 546-nm
excitation filter and a 590-nm absorption filter for PI. Healthy cells
were unstained by either annexin V or PI, early apoptotic cells were
stained only by annexin V, and dead cells were stained by annexin V and
PI. The assay was repeated with three separate granulosa cell
preparations.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
;
P < 0.05). There was no difference in cell numbers
between wells treated with IgG in the absence or presence of IFN
(6.9 ± 0.6 x 105 vs. 7.9 ±
0.4 x 105 cells/well, respectively; P
> 0.05).
|
).
|
; P
< 0.05). TNF treatment in the presence of CX had no effect alone but
potentiated the effect of Fas mAb, resulting in 56% killing in the
absence of IFN and 86% killing in the presence of IFN
(P < 0.05). Pretreatment with IFN had no effect on
cell numbers in IgG-treated wells (4.3 ± 1.2 x
105 cells/well without IFN vs. 4.6 ±
1.2 x 105 cells/well with IFN; P >
0.05). Wells treated with CX and IgG in the absence and presence of IFN
had 19% and 27% fewer cells, respectively, than wells treated with
IgG alone in the absence or presence of IFN, but the
difference was not significant (P > 0.05).
|
).
Cultures that were also treated with CX contained a greater percentage
of cells with labeled nuclei and were sparse due to the detachment of
dead cells from the culture dish (Fig. 4c). Cultures treated with IFN,
TNF, and CX in the absence of Fas mAb did not show significant staining
of nuclei (Fig. 4a).
|
, b and c),
indicating that these cells were expressing phosphatidylserine on the
outer membrane. Cells treated with IFN, TNF, and Fas mAb in the
presence of CX had numerous cells stained with annexin V but not with
PI (Fig. 5, e and f). The low number of cells staining with PI is due
to the failure of dead cells to adhere to the plate. Control cells
treated with IFN and TNF in the presence of CX show little staining
with either annexin V or PI (Fig. 5, h and i).
|
).
Therefore, regardless of whether TNF was given as a pretreatment (Fig. 6A) or simultaneously with Fas mAb (Fig. 1
), stimulation of granulosa
cells with both IFN and TNF was necessary for Fas-mediated killing.
There was no effect of pretreatment on cell numbers in control
(IgG-treated) wells. Cell counts were 5.8 ± 1.5 x
105, 6.3 ± 1.5 x 105, 6.4 ±
1.7 x 105, and 6.1 ± 1.0 x
105 cells/well in control and TNF-, IFN-, and TNF- plus
IFN-pretreated wells, respectively.
|
). Fas antigen mRNA was
detectable in control cultures and was 3.2-fold higher in cultures
treated with IFN (P > 0.05 vs. control),
3.5-fold higher in cultures treated with TNF (P <
0.05), and increased 19-fold in cultures treated with the combination
of IFN and TNF (P < 0.01).
Immunohistochemistry was used to assess the expression of Fas antigen
protein in cultured granulosa cells. Positive staining for Fas antigen
was detected in untreated granulosa cells. Treatment with TNF or IFN
alone and with TNF plus IFN resulted in increased positive staining for
Fas antigen (Fig. 7).
|
). These results suggest that
the effects of mouse TNF on Fas antigen expression and Fas-mediated
death are transmitted through TNFRII.
|
).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A potential role for TNF in modulating Fas-mediated killing in the ovary is consistent with its recognized role as a modulator of a wide variety of ovarian functions (19, 33). Production of TNF by ovarian macrophages, endothelial cells, granulosa cells, thecal cells, and oocytes has been demonstrated in a number of species (19), including the mouse (34). Effects of TNF on cultured ovarian cells include inhibition of gonadotropin-stimulated steroidogenesis (35, 36, 37, 38), of FSH-induced formation of LH receptors by granulosa cells (39), and of LH-induced androgen production by thecal cells (40). These studies reported no effects of TNF on cell viability or cell number. However, TNF was reported to induce proliferation of human granulosa/luteal cells (41, 42) and was cytotoxic to bovine luteal cells that were cotreated with IFN for 7 days (43). Two recent studies reported that TNF induced apoptosis of granulosa cells. TNF inhibited the effect of FSH to suppress the spontaneous onset of DNA fragmentation in small antral rat follicles cultured in serum-free medium (3). TNF promoted oligonucleosome formation in granulosa cells from apoptosis-susceptible (prehierarchal) hen follicles, but not in those from apoptosis-resistant follicles (4). Variable effects of TNF on cell viability observed in different studies may be due to differences in species, stage of cellular differentiation, culture conditions, doses of TNF, and assays for viability. In our experiments, TNF alone and TNF in the presence of IFN had no effect on cell viability. However, combined treatment with IFN and TNF potentiated Fas-mediated killing.
Mouse TNF increased Fas antigen expression and in combination with IFN potentiated Fas-mediated apoptosis, whereas hTNF had no effect. As hTNF interacts with mouse TNFRI but not mouse TNFRII (26), these results suggest that effects of mouse TNF on Fas-mediated apoptosis are mediated by TNFRII. Cytotoxic effects of TNF have generally been attributed to interactions with TNFRI, which contains a cytoplasmic death domain involved in signal transduction (44), although an accessory role for TNFRII in cytotoxicity has been observed in some cell types (45). The effects of TNF on cell proliferation are thought to be mediated through TNFRII (46). TNFRII is linked to a signaling pathway involving TRAF proteins (TNF receptor-associated factors). Recent studies have revealed significant interactions between TNFRI and TNFRII signaling pathways that may help to explain both distinct and overlapping effects mediated by the receptors (47). Ovarian expression of TNF receptors has not been examined extensively. However, human cumulus cells and oocytes were reported to express TNFRII but not TNFRI (48), and expression of mRNA for both receptors was detected in whole rat ovaries (49).
IFN is found in follicular fluid (50), and a possible source is lymphocytes that infiltrate the ovary (33). IFN modulates steroidogenesis and differentiative functions of ovarian cells in vitro (51, 52, 53, 54, 55). It remains to be determined whether IFN and TNF are physiological regulators of Fas in the ovary and whether other factors in the ovary modulate Fas-mediated killing.
The ability of CX to potentiate Fas-mediated killing suggests that granulosa cells have inhibitors of the Fas pathway. In the presence of CX, Fas mAb treatment alone induced apoptosis. Therefore, the level of Fas antigen expression by granulosa cells is sufficient for stimulation of the Fas pathway when inhibitory proteins are removed by treatment with CX. In a number of other cell types, treatment with CX or actinomycin D induced susceptibility to Fas-mediated apoptosis (56, 57, 58). TNF promoted Fas mAb-induced killing in the presence and absence of CX. CX would be expected to block induction of Fas antigen expression by TNF and to prevent the production of protein inhibitors of the Fas pathway. Therefore, it is unlikely that TNF potentiates Fas- mediated killing solely by increasing Fas expression or reducing expression of inhibitors of the Fas pathway.
FAP-1 is a protein tyrosine phosphatase that binds to the cytoplasmic death domain of the Fas antigen and inhibits Fas-mediated killing. FAP-1 is expressed at higher levels in cell lines that are resistant to Fas-mediated killing (23). IFN and TNF had no effect on FAP-1 mRNA expression by granulosa cells, suggesting that potentiation of Fas-induced killing by TNF and IFN is not mediated by alterations in FAP-1 mRNA expression. However, these results do not rule out the possibility that IFN and TNF may alter FAP-1 activity. Although not tested directly in this study, potentiation of Fas-mediated killing by CX may be due to removal of inhibitory proteins such as FAP-1. Recently, a protein known as FLIP (FLICE-inhibitory protein) was identified that prevents apoptosis initiated by death receptors such as Fas antigen, TNF receptor, and TRAMP (DR3/wsl/Apo-3) by interacting with the adaptor protein FADD and the caspace FLICE (24). A family of apoptosis inhibitors, the IAPs, have been identified and are expressed in the ovary (59, 60). Further research is needed to determine the identity and role of specific inhibitors of Fas-mediated apoptosis in ovarian cells.
Considerable variability exists in reports of cell types that express Fas antigen and Fas ligand in the ovary. Fas antigen was detected by immunohistochemistry in the granulosa cell layer of all but small follicles in mice (61). Immunoreactive Fas antigen was higher in granulosa cells from atretic follicles than in those from healthy follicles in humans and rats (13, 14, 15). Contradictory results suggest that oocytes do [human (14), rat (12)) or do not (human (11), rat (13, 15), mouse (61)] express Fas antigen. Studies in the human (11, 14) and rat (15) suggest that Fas antigen is expressed in the theca during certain stages of development. FasL expression was detected by immunohistochemistry in oocytes, but not in other ovarian cells of rats (13), but was detected by immunohistochemistry, RT-PCR, and in situ hybridization in the granulosa cell layer and not other follicular sites in mice (16) and in granulosa and thecal cell layers in rats (15). Additional studies are likely to reconcile differences in reports on the cellular sites of Fas antigen and FasL expression in the ovary. The results of our study confirm the expression of Fas antigen in mouse granulosa cells and indicate that factors in addition to Fas antigen expression and availability of FasL are required for Fas-mediated apoptosis.
The Fas-mediated pathway of apoptosis is functional in mouse granulosa cells that are stimulated with IFN and TNF. These cytokines may function at least partially by increasing Fas antigen expression. Granulosa cells appear to have inhibitors of the Fas antigen pathway, since treatment with CX potentiates Fas-mediated death. TNF promotes Fas-mediated killing and remains effective when protein synthesis is inhibited by CX. This suggests that TNF does not act simply by increasing Fas antigen expression or by decreasing protein inhibitors of the Fas pathway, as both processes would not occur in the presence of CX. To understand the physiological role of the Fas antigen pathway in ovarian cell apoptosis, further information is needed on cellular sites of Fas antigen and FasL expression and on factors that regulate activation of the Fas-mediated death pathway.
|
Footnotes |
|---|
Received March 27, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and its second messenger, ceramide, stimulate
apoptosis in cultured ovarian follicles. Endocrinology 137:4864–4870[Abstract]
in follicular development, ovulation and the life span of
the corpus luteum. Dom Anim Endocrinol 14:1–15[CrossRef][Medline]
gene expression in
mouse oocytes and follicular cells. Biol Reprod 48:707–714[Abstract]
inhibits gonadotropin hormonal action in
nontransformed ovarian granulosa cells. J Biol Chem 264:11591–11597 inhibits gonadotropin-supported protesterone
accumulation by differentiating and luteinized murine granulosa cells.
Am J Obstet Gynecol 162:889–899[Medline]
inhibits follicle-stimulating hormone-induced
differentiation in cultured rat granulosa cells. Biochem Biophys Res
Commun 163:1038–1046[CrossRef][Medline]
attenuation of luteinizing hormone-stimulated
androstenedione production by ovarian theca-interstitial cells:
inhibition at loci within the adenosine 3',5'-monophosphate-dependent
signaling pathway. Endocrinology 133:2269–2276[Abstract]
in the human ovary: presence in follicular
fluid and effects on cell proliferation and prostaglandin production.
Fertil Steril 58:934–940[Medline]
alters steroidogenesis and
stimulates proliferation of human ovarian granulosal cells in
vitro. Fertil Steril 59:332–338[Medline]
alters bovine luteal cell synthetic capacity and viability.
Endocrinology 130:854–860[Abstract]
B activation.
Cell 81:495–504[CrossRef][Medline]
and its receptors type I and type II in human
oocytes. Mol Reprod Dev 47:127–133[CrossRef][Medline]
, tumor necrosis factor ,
and interleukin-1 in modulating progesterone and oestradiol production
by human luteinized granulosa cells in culture. Hum Reprod 7:1361–1364. J Endocrinol 133:131–139. Biol Reprod 44:357–363[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  X. Y. Wang, J. G. Crowston, H. Zoellner, and P. R. Healey Interferon-{alpha} and Interferon-{gamma} Sensitize Human Tenon Fibroblasts to Mitomycin-C Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3655 - 3661. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A L Johnson, C. Ratajczak, M. J Haugen, H.-K. Liu, and D. C Woods Tumor necrosis factor-related apoptosis inducing ligand expression and activity in hen granulosa cells Reproduction, March 1, 2007; 133(3): 609 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Greenfeld, K. F. Roby, M. E. Pepling, J. K. Babus, P. F. Terranova, and J. A. Flaws Tumor Necrosis Factor (TNF) Receptor Type 2 Is an Important Mediator of TNF alpha Function in the Mouse Ovary Biol Reprod, February 1, 2007; 76(2): 224 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Liptak, B. T. Sullivan, L. E. Henkes, M. P.B. Wijayagunawardane, A. Miyamoto, J. S. Davis, B. R. Rueda, and D. H. Townson Cooperative Expression of Monocyte Chemoattractant Protein 1 Within the Bovine Corpus Luteum: Evidence of Immune Cell-Endothelial Cell Interactions in a Coculture System Biol Reprod, May 1, 2005; 72(5): 1169 - 1176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Chen, T. Yano, H. Matsumi, Y. Osuga, N. Yano, J. Xu, O. Wada, K. Koga, T. Fujiwara, K. Kugu, et al. Cross-Talk between Fas/Fas Ligand System and Nitric Oxide in the Pathway Subserving Granulosa Cell Apoptosis: A Possible Regulatory Mechanism for Ovarian Follicle Atresia Endocrinology, February 1, 2005; 146(2): 808 - 815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.T. Bridgham and A.L. Johnson Alternatively Spliced Variants of Gallus gallus TNFRSF23 Are Expressed in the Ovary and Differentially Regulated by Cell Signaling Pathways Biol Reprod, April 1, 2004; 70(4): 972 - 979. [Abstract] [Full Text] [PDF]
|
|
