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Regular Article |
a Department of Animal Science, Cornell University, Ithaca, New York 14853
ABSTRACT
Our previous studies have shown that bovine granulosa cells cultured in basal media supplemented with 5% fetal bovine serum (BM-FBS) are resistant to apoptosis induced by recombinant Fas ligand (FasL) unless pretreated with interferon-
(IFN). Experiments were conducted to test the hypothesis that serum and growth factors alter the susceptibility of granulosa cells to FasL-induced apoptosis. Granulosa cells were cultured in BM-FBS, BM containing insulin, transferrin, selenium, and BSA (BM-ITS), and in BM-ITS supplemented with insulin-like growth factor-I (IGF). Cells were susceptible to FasL-induced killing in BM-ITS (27% killing) but were resistant in BM-FBS and in BM-ITS containing IGF (P < 0.05 vs. killing in BM-ITS). Exposure of phosphatidylserine residues on the outer cell membrane, an early marker of apoptosis, was stimulated by FasL and prevented in the presence of IGF. Neutralization of IGF activity in serum with IGF binding protein 3 reduced the protective effect of FBS on FasL-induced killing (P < 0.05), suggesting that IGF is an inhibitory component in FBS. Cotreatment with IFN overcame the inhibitory effects of serum and IGF on FasL-induced killing (31% and 29% killing, respectively, P > 0.05), but IFN did not potentiate killing of cells cultured in BM-ITS. IFN increased expression of Fas antigen (Fas, the receptor for FasL) mRNA five- to sevenfold (P < 0.05) and increased immunostaining for Fas protein similarly in all types of media. Addition of the growth factors epidermal growth factor or basic fibroblast growth factor to BM-ITS also inhibited FasL-induced killing (P < 0.05), whereas keratinocyte growth factor, transforming growth factor, platelet-derived growth factor, FSH, and LH had no effect. In summary, FasL-induced killing is inhibited by FBS and certain growth factors. IFN increased expression of Fas similarly in all types of media but was required for FasL-induced killing only in BM containing FBS or IGF. Therefore, modulation of responsiveness to FasL-induced apoptosis by growth factors and IFN is not directly related to the level of Fas expression.
apoptosis, cytokines, granulosa cells, growth factors, ovary
INTRODUCTION
The death of granulosa cells associated with follicular atresia occurs through the process of apoptosis [1]. The signals that induce granulosa cells to undergo apoptosis have not been completely defined but may include lack of growth factors and/or induction by cytotoxic stimuli such as tumor necrosis factor (TNF) or the Fas ligand (FasL) [2]. FasL is a cell surface molecule belonging to the TNF family that induces apoptosis in sensitive cells by ligation to its receptor, Fas antigen (Fas). Binding of the adapter protein, FADD, to the cytoplasmic "death domain" of Fas activates a cascade of cysteine proteases (caspases) that cleave key cellular substrates resulting in cell death. In addition, binding of a protein, DAXX, to the death domain of Fas mediates apoptosis by activating the stress-activated c-Jun NH2-terminal kinase (JNK) pathway [3]. Granulosa cells express Fas mRNA and protein, and Fas expression is elevated in atretic follicles of the rat, human, and cow [4–8]. Messenger RNA and protein for FasL are also expressed in the ovary, and expression is elevated in atretic follicles [5, 6, 9]. Our studies have shown that under certain conditions bovine and murine granulosa cells and human granulosa/luteal cells undergo apoptosis when treated with soluble FasL or agonistic anti-Fas antibodies in vitro [8, 10–12]. In human and bovine granulosa cells, pretreatment or cotreatment with interferon- (IFN) was necessary for Fas-mediated apoptosis to occur [8, 10, 12], while in mouse granulosa cells pretreatment with IFN and TNF was required [11]. In all of these studies granulosa cells were cultured in media containing 5% fetal bovine serum (FBS). Fetal bovine serum contains growth factors, some of which have been shown to regulate follicular growth and atresia [1, 13]. In the present study, FasL-induced killing was examined in granulosa cells cultured in defined media in the presence or absence of various growth factors and in media containing serum. The results show that granulosa cells cultured in defined media are susceptible to FasL-induced apoptosis in the absence and presence of IFN and that serum, insulin-like growth factor-I (IGF), and a number of other growth factors inhibit FasL-induced killing by a mechanism independent of the level of Fas expression. These results are relevant to the situation in vivo in which the presence of growth factors within healthy follicles may be necessary to inhibit Fas-mediated apoptosis and promote follicle development.
MATERIALS AND METHODS
Materials
All culture media reagents were obtained from Gibco BRL (Grand Island, NY), as were recombinant human basic fibroblast growth factor (bFGF), murine epidermal growth factor (EGF), recombinant human IGF, recombinant human platelet-derived growth factor-BB (PDGF), and recombinant human transforming growth factor ß1 (TGF). Soluble recombinant human FasL, recombinant human IGF binding protein 3 (IGF-BP3), mouse monoclonal anti-human Fas (clone CH-11), and recombinant human keratinocyte growth factor (KGF) were obtained from Upstate Biotechnology (Lake Placid, NY). Cy2-conjugated goat anti-mouse IgM was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Tissue culture plates were obtained from Corning-Costar (Cambridge, MA), except that Slide-well chambers were from Nunc-Intermed (Naperville, IL). Avian myeloblastosis virus reverse transcriptase (RT) was obtained from Promega (Madison, WI), random hexamer from Pharmacia (Piscataway, NJ), and Taq polymerase from Fisher (Pittsburgh, PA). Bovine IFN was graciously provided by Dr. Dale Godson, Veterinary Infectious Disease Organization (Saskatoon, Saskatchewan, Canada). Ovine FSH (lot no. NIDDK-oFSH-20) and ovine LH (oLH; lot no. NIDDK-oLH-26) were obtained from Dr. A.F. Parlow, Harbor-UCLA Medical Center.
Cell Culture
Freshly excised cow ovaries were obtained from an abattoir, transported in saline at room temperature (approximately 1.5 h), and processed immediately thereafter. Granulosa cells were obtained by aspiration of 5–10-mm follicles with a 16-gauge needle, followed by flushing of the follicles with Dulbecco modified Eagle medium (DMEM) containing 0.002 M EDTA. Cells were collected by centrifugation, washed, counted, and plated in basal media (BM; DMEM-F12 medium supplemented with 1 mM pyruvate, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone) containing 10% FBS on Day 0. Cells were plated at approximately 5 x 104 cells/well in 96-well plates for analysis of FasL-induced apoptosis, or 0.8–1 x 106 cells/well in 6-well plates for analysis of RNA. For cytochemical analyses, cells were plated at 4 x 104 cells/well in 8 x 8-mm slide-well chambers. Medium was replaced on Day 1. On Day 2, medium was replaced with either BM containing 5% FBS (BM-FBS) or BM containing 100 ng/ml insulin, 5 µg/ml transferrin, 20 nM selenium, and 0.1% BSA (BM-ITS). The concentration of insulin in BM-ITS is significantly lower than commercial preparations of ITS. On Day 3, cells were treated in the same media with 0 or 100 U/ml IFN and with 0 or 100 ng/ml FasL. The IFN dose was based on preliminary experiments and is the minimum dose required to maximally stimulate FasL-induced killing. On Day 4, cells were incubated 6–8 min with a solution of 0.25% trypsin-1 mM EDTA, trypan blue was added to a final concentration of 0.2%, and live cells excluding trypan blue counted within 4 min in a hemacytometer. Additional cultures were used to examine the effects of media and IFN on expression of Fas mRNA. Cells were cultured as described above on Days 0–2 and treated on Day 3 with 0 or 100 U/ml IFN in either BM-FBS or BM-ITS. On Day 4, cells were frozen at -80°C for subsequent analysis of Fas mRNA.
In order to test effects of IGF on Fas-mediated killing, cells were cultured in BM containing 10% FBS on Days 0 and 1, and on Day 2 in BM-ITS containing 0 or 100 ng/ml IGF. The IGF concentration was established in preliminary experiments as the minimum dose required to block FasL-induced killing in ITS. On Day 3, cultures were treated in the same media with 0 or 100 U/ml IFN and 0 or 100 ng/ml FasL. On Day 4, viable cells were counted as described above. Additional cultures were used to examine the effects of IGF and IFN on expression of Fas mRNA. Cells were cultured on Days 0 and 1 in BM containing 10% FBS and on Day 2 in BM-ITS containing 0 or 100 ng/ml IGF. On Day 3 cells were cultured in BM-ITS containing 0 or 100 ng/ml IGF and 0 or 100 U/ml IFN. On Day 4, cells were frozen at -80°C for subsequent analysis of Fas mRNA.
Initial experiments showed that the presence of 5% FBS in BM inhibited FasL-induced killing. An experiment was performed to test the effects of neutralizing IGF activity in FBS using IGF-BP3. The IGF-BP3 was used previously to inhibit effects of IGF-1 on rat granulosa cells [14]. Preliminary studies showed that 1.25% FBS was the minimal concentration required to completely block FasL-induced killing. Therefore, in subsequent experiments 2% FBS was used instead of 5% FBS in order to increase the effectiveness of IFG-BP3 to neutralize IGF activity. Cells were cultured on Days 0 and 1 in BM containing 10% FBS. On Day 2, medium was changed to BM containing 2% FBS. On Day 3, cells were treated with 0 or 100 ng/ml FasL in BM-2% FBS containing 0 or 62.5 U/ml IGF-BP3. On Day 4, cells were counted as described above.
Effects of growth factors and gonadotropins on FasL-induced killing were determined. Cells were cultured in BM containing 10% FBS on Days 0 and 1, and on Day 2 in BM-ITS alone or BM-ITS supplemented with various growth factors (100 ng/ml IGF, 50 ng/ml EGF, 50 ng/ml bFGF, 50 ng/ml KGF, 10 ng/ml TGF, and 10 ng/ml PDGF) or gonadotropins (2 and 100 ng/ml FSH and 2 and 100 ng/ml LH). On Day 3, cultures were treated with 0 or 100 ng/ml FasL in BM-ITS media with or without the various growth factors or gonadotropins. On Day 4, viable cells were counted as described above. The doses of growth factors and gonadotropins used are based on previously reported effective doses of growth factors in bovine ovarian cells (EGF [15, 16], bFGF [15], KGF [17], TGF [15, 18], PDGF [19] and LH and FSH at high and low doses as described [20]).
Analysis of Fas mRNA
Messenger RNA for Fas was quantified by a competitive RT polymerase chain reaction (RT-PCR) assay as described previously [10]. The RNA was reverse transcribed in the presence of various concentrations of an internal RNA standard. The internal RNA standard was prepared by in vitro transcription of mutated bovine Fas cDNA containing a 50-base pair (bp) deletion internal to the PCR primer binding sites. Complementary DNA in the RT reaction was amplified by PCR in the presence of 32P-dCTP. Primers were designed to span the positions of three introns in order to distinguish amplification of cDNA from contaminating genomic DNA. Amplification resulted in a 206-bp fragment for the wild-type Fas RNA and a 156-bp fragment for the internal RNA standard. The RT-PCR products were fractionated on a 2% agarose gel. The gel was dried and radioactive signal was quantified on a Fuji BAS1000 phosphoimager. The concentration of Fas mRNA in each sample was calculated by regression of the log (sample signal/standard signal) versus log standard concentration. Samples from the same culture preparation were assayed together along with an RNA pool and water blank. The between-assay coefficient of variation was 12.6%.
Immunocytochemistry for Fas
Protein expression for Fas was detected on fixed cultured cells using a mouse monoclonal IgM antibody against the extracellular portion of human Fas (clone CH-11). This antibody was shown to react with bovine Fas overexpressed in L929 cells and with MDBK cells (a bovine kidney cell line) [21]. As a positive control, MDBK cells were cultured with or without 100 U/ml IFN. Interferon- has been shown to increase Fas expression in a variety of cell types [11]. Cells were fixed in acetone for 2 min at -20°C, rinsed, and blocked with PBS-2% normal goat serum (NGS). Cells were incubated in PBS-2% NGS containing 2 µg/ml anti-Fas antibody or mouse IgM to assess nonspecific binding, at 37°C for 1 h. Two micrograms per milliliter of Cy2-conjugated goat anti-mouse IgM was used for fluorescent detection of the anti-Fas antibody. Cells were observed under phase-contrast and epifluorescent illumination using a 495-nm excitation filter and a 520-nm absorption filter. Images were obtained with a Spot II charge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI). Immunocytochemistry for Fas was performed on three separate granulosa cell preparations.
Detection of Membrane-Associated Phosphatidylserine
The translocation of phosphatidylserine from the inside to the outside of the cell membrane is a unique and early marker for apoptosis that can be detected by binding of annexin V [22]. Staining of cells with Alexa 488-conjugated annexin V and propidium iodide (PI) were performed using the Vybrant apoptosis assay kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions as described previously [10]. Healthy live cells do not stain with either annexin V or PI, cells in early stages of apoptosis stain positively for annexin V only, while dead and dying cells with permeable membranes stain positively for both PI and annexin V. The assay was repeated on three separate granulosa cell preparations.
Statistical Analysis
Data were analyzed by a randomized complete block ANOVA [23]. Duncans new multiple range test was used for comparison of means when overall significance was observed. The mRNA concentration was log transformed prior to analysis.
RESULTS
Effects of Defined Versus Serum-Supplemented Media on FasL-Induced Killing and Fas Expression
No significant killing was induced by FasL in granulosa cells cultured in BM-FBS, whereas cotreatment with IFN and FasL induced 31% killing (Fig. 1). In cells that were plated in media containing serum and then cultured in the absence of serum (BM-ITS), treatment with FasL alone induced 27% killing and cotreatment with FasL and IFN also induced 27% killing (Fig. 1). The BM-FBS control cultures (no FasL) had 7.0 ± 0.5 x 104 cells/well on Day 4 of culture, 28% greater than the number of cells plated on Day 0 (5.5 ± 0.5 x 104 cells/well). There were 5.8 ± 0.7 x 104 cells/well in BM-ITS control cultures on Day 4, less than in BM-FBS control cultures (P < 0.05). Treatment with IFN alone had no effect on viability of cells cultured in either BM-FBS or BM-ITS (not shown). The concentration of Fas mRNA was similar in granulosa cells cultured in BM-FBS and BM-ITS and was increased sevenfold by IFN in cells cultured in both types of media (Fig. 1).
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Effects of IGF on FasL-Induced Killing and Fas Expression
Addition of IGF to cells cultured in BM-ITS prevented the cytotoxic effect of FasL (Fig. 2). Interferon- had no effect on FasL-induced killing in granulosa cells cultured in BM-ITS but overcame the inhibitory effect of IGF on FasL-induced killing (Fig. 2). There were significantly more cells in control cultures (no FasL) containing IGF compared to the number of cells in cultures containing BM-ITS alone (8.2 ± 2.9 x 104 cells/well vs. 4.7 ± 1.6 x 104 cells/well on Day 4 of culture, respectively; P < 0.05). The number of cells plated on Day 0 was 5.5 ± 0.4 x 104 cells/well), and IFN treatment alone had no effect on cell numbers in either media. IGF had no effect on the concentration of Fas mRNA, but IFN induced a 4.8-fold increase in cells cultured in BM-ITS and a 5.8-fold increase in cells cultured in BM-ITS supplemented with IGF (Fig. 2).
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Immunocytochemical staining showed that Fas protein was expressed similarly in cells cultured in BM-FBS, BM-ITS, and BM-ITS supplemented with IGF and was increased similarly by addition of IFN to each type of media (Fig. 3).
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Binding of annexin V to phosphatidylserine on the outer cell membrane was used to assess qualitatively whether cell death occurred by apoptosis. Cells were treated with or without IGF and with or without FasL in BM-ITS exactly as described above and annexin binding assessed 12 h after treatment with FasL when cells were actively dying. In cultures treated with FasL alone, increased numbers of cells were stained with annexin V but not PI, consistent with apoptosis. Minimal apoptotic cells were observed in cultures treated with IGF + FasL or in control cultures not treated with FasL (Fig. 4).
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Experiments were performed to determine whether inhibitory effects of FBS on FasL-induced killing are mediated by IGF present in FBS. Preliminary experiments (not shown) showed that 1.25% FBS in medium was the minimal concentration that completely blocked FasL-induced killing of granulosa cells Therefore, the effect of using IGF-BP3 to neutralize IGF activity on FasL-induced killing of cells cultured in 2% FBS was tested. No cytotoxicity was observed in FasL-treated cells cultured in BM containing 2% FBS, but FasL did induce 15.8% killing when cells were cotreated with IGF-BP3 (Fig. 5). Treatment with IGF-BP3 alone had no effect on cell viability.
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Effect of Growth Factors and Gonadotropins on FasL-Induced Killing
The effects of a panel of growth factors and gonadotropins on granulosa cell numbers and response to FasL-induced killing was investigated. Addition of IGF, EGF, and bFGF to BM-ITS media inhibited FasL-induced killing (Fig. 6, P < 0.05 vs. BM-ITS). KGF, TGF, PDGF, and both high and low doses of the gonadotropins FSH and LH had no effect on FasL-induced killing. Addition of IGF, EGF, or bFGF to BM-ITS in the absence of FasL also increased granulosa cell numbers relative to cultures in BM-ITS alone (P < 0.05), while the other growth factors and the gonadotropins had no significant effect on granulosa cell numbers (Fig. 6).
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DISCUSSION
The data demonstrate that granulosa cells cultured in media containing FBS, which has significant IGF activity [24], required treatment with IFN for Fas-mediated killing. Cells cultured in defined media containing ITS died in response to FasL in equal numbers with or without IFN treatment. However, when 100 ng/ml IGF was added to the defined media, granulosa cells did not die in response to FasL unless also treated with IFN, mimicking the results observed in cells cultured in media containing serum. When cells cultured in media containing serum were treated with FasL in the presence of IGF-BP3 that neutralizes IGF activity killing was restored. This strongly suggests that IGF is a factor in serum-containing media that blocks Fas-mediated killing. The fact that IGF inhibits FasL-induced killing in vitro is relevant to the physiology of follicle development. Growth factors produced within the follicle, such as IGF, are proposed to promote growth, differentiation, and survival of follicular cells [2, 25, 26]. The IGF levels in follicular fluid of bovine follicles were positively correlated with estradiol concentrations and follicular diameter in several but not all studies. In addition, a number of studies reported that bioavailability of IGF-1 to granulosa cells may be reduced by elevated levels of low molecular weight IGF-binding proteins in follicular fluid of atretic follicles relative to that observed in healthy follicles [26]. One of the mechanisms whereby IGF promotes follicle survival may be inhibition of the Fas pathway.
The fact that IGF decreased annexin V staining of cells treated with FasL indicates that IGF inhibited apoptosis and that effects of IGF to maintain the number of viable cells was not simply due to a stimulatory effect on cell proliferation. Annexin V staining is useful for detecting the early stages of apoptosis, when phosphatidylserine residues are exposed on the outer membrane but cell membranes are intact and the vital dye, PI, is excluded. At later stages of apoptosis, cells stain positively for both annexin V and PI and cannot be distinguished from necrotic cells [22]. Granulosa cells become detached from the culture dish as apoptosis proceeds and are then not available for staining with annexin V. Accordingly, counting the number of viable cells remaining on plates at 24 h after treatment with FasL was used to quantify the total number of cells lost from cultures. Annexin V staining at 12 h of culture was used to assess qualitatively whether cells died by apoptosis. Although flow cytometry has been used to quantify annexin V staining in nonadherent cell types, this was not attempted in the present study because of concerns that removal of adherent cells from the culture dish would affect membrane staining.
Additional growth factors besides IGF have been identified in ovarian follicles and have been implicated in promoting survival of granulosa cells in vitro [27] and during follicle development [2, 13]. EGF and bFGF also inhibited killing by FasL in BM-ITS, while other growth factors (KGF, TGF, and PDGF) and the gonadotropins FSH and LH had no effect. Whereas FSH and IGF prevented spontaneous apoptosis of pig granulosa cells cultured in a limiting concentration of serum [28], IGF did not protect rat granulosa cells cultured in serum-free medium [27]. Interestingly, only those growth factors that caused a significant increase in cell number relative to BM-ITS were effective in blocking FasL-induced killing. One possibility is that granulosa cells may be susceptible to apoptosis at particular stages of the cell cycle and that the fraction of cells at this stage may be highest in the absence of growth factors. An interaction between the cell cycle and susceptibility to apoptosis has been demonstrated in other cell types [29]. The fact that only 30% of granulosa cells were killed by FasL in BM-ITS or in BM containing serum or IGF in the presence of IFN suggests that granulosa cells within cultures may be in different stages of the cell cycle and/or at different stages of differentiation. IFN has been shown to modulate the cell cycle in other cell types [30]. In previous studies we found that granulosa cells from preovulatory bovine follicles isolated about 12 h after the LH surge are completely resistant to Fas-mediated killing in vitro [8]. Therefore, differentiation of granulosa cells is likely to alter susceptibility to apoptosis. Future experiments will be necessary to address these possibilities.
Another conclusion that can be drawn from these studies is that the responsiveness of granulosa cells to FasL is to some extent independent of the amount of Fas expressed. Cells treated with FasL in media containing FBS or IGF expressed as much Fas mRNA and apparently as much Fas protein as cells treated with FasL in BM-ITS, yet only the cells treated with FasL in BM-ITS underwent apoptosis. IFN increased expression of Fas mRNA and protein similarly in all the media tested, but while IFN increased FasL-induced killing in media containing FBS or IGF, it had no effect on killing by FasL in BM-ITS. Therefore, IFN appears to be necessary to overcome inhibitory effects of serum and IGF on FasL-induced killing, and this effect is not directly related to increased expression of Fas. Although several reports have shown changes in expression of Fas in the follicle consistent with a role for Fas-mediated killing in follicular atresia [4–8], the data presented here demonstrate that the level of expression of Fas is not a clear indicator of Fas responsiveness. It is likely that modulation of the responsiveness of the Fas pathway by growth factors is a critical element in regulating follicular atresia.
A number of growth factors including IGF, EGF, and bFGF have been shown to promote cell survival by stimulating the activity of phosphoinositide 3-OH kinase that in turn phosphorylates and activates the kinase, Akt-1 [31]. The Akt-1 pathway has been shown to mediate the positive effects of IGF on survival of pig granulosa cells [32]. Proteins that are phosphorylated and inactivated by Akt-1 include the proapoptotic proteins Bad and caspase-9, as well as the transcription factor, forkhead, that stimulates transcription of FasL [33]. Aberrant overactivation of the Akt-1 pathway in mice prevented Fas-mediated apoptosis [34]. PDGF also stimulates the Akt-1 pathway in many cell types [31], but it had no effect on FasL-induced killing of granulosa cells. PDGF modulates growth and differentiation of cultured rat granulosa cells [35], but porcine granulosa cells lack PDGF receptors [19], and it is not currently known whether bovine granulosa cells express PDGF receptors.
In summary, granulosa cells are resistant to FasL-induced apoptosis in the presence of serum and various growth factors. IFN overcomes the inhibitory effects of serum and IGF on FasL-induced killing, and this effect is not simply related to increased levels of Fas expression. It is likely that responsiveness of granulosa cells to Fas-mediated killing in vivo is dependent upon the complement of survival factors within the follicle.
ACKNOWLEDGMENTS
The authors thank Dr. Dale Godson for providing bovine IFN.
FOOTNOTES
1 This work was supported by grants from the National Institutes of Health (HD 32535) and U.S. Department of Agriculture (98-35203-6220). 
2 Correspondence: Susan M. Quirk, 258 Morrison Hall, Cornell University, Ithaca, NY 14853. FAX: 607 255 9829; smq1{at}cornell.edu
Accepted: June 6, 2000.
Received: April 14, 2000.
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