| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Animal Science, Cornell University, Ithaca, New York 14853
Address all correspondence and requests for reprints to: Susan M. Quirk, Department of Animal Science, Morrison Hall, Cornell University, Ithaca, New York 14853. E-mail: smq1{at}cornell.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
IGF-I is critical for ovarian follicle development. Genetically engineered mice that lack IGF-I are infertile. Follicle development is arrested at the small antral stage, and mature Graafian follicles are not produced (11, 12). In cattle, rodents, and humans, healthy follicles differ from atretic follicles in having reduced levels of inhibitory IGF-binding proteins, and this promotes increased bioavailability of IGF (13, 14, 15, 16). IGF-I stimulates two major signal transduction pathways: the phosphoinositide 3'-OH kinase (PI3K) pathway and the MAPK pathway (also known as the ERK pathway) (17). Binding of IGF-I to its receptor initiates events leading to recruitment of PI3K to the inner surface of the plasma membrane where it catalyzes production of 3'-phosphorylated phosphoinositides. These phospholipids bind to Akt kinase, leading to its relocation to the plasma membrane and phosphorylation by regulatory kinases. Akt that has been activated by phosphorylation regulates multiple cellular processes. Activation of the MAPK pathway by IGF-I requires activation of Ras and is mediated by a cascade of successive protein phosphorylation reactions involving Raf, MAPK kinase (MEK-1 and -2) and finally MAPK/ERK-1 and -2. Depending upon the cell type, activation of the PI3K or the MAPK pathway by IGF-I has been reported to stimulate proliferation, differentiation, and/or cell survival (16, 17, 18, 19).
IGF-I stimulates proliferation as well as survival in a number of cell types, suggesting potential interactions between these processes. Mounting evidence suggests that regulation of apoptosis and of the cell cycle are tightly linked (20, 21, 22, 23). Common control of these two processes could contribute to maintaining the balance between cell proliferation and cell death. Cell cycle progression is mediated by cyclin dependent kinases (cdks) (24). cdks Are activated by binding to specific cyclin proteins that are synthesized periodically during the cell cycle. Progression from G1 to S phase of the cell cycle requires formation of complexes between cdk4 or cdk6 and D-type cyclins during early to mid-G1, followed by formation of complexes of cdk2 and cyclin E during late G1. Accumulation of the D-type cyclins in G1 is required for cell cycle entry and is regulated by extracellular growth factors. Enzymatic activity of cyclin/cdk complexes is regulated by cdk inhibitors belonging to the INK4 and Cip/Kip families.
In the studies presented here, we examine the mechanism of IGF-I-mediated protection against apoptosis in bovine granulosa cells and its relationship to cell cycle control. We show that IGF-I protects granulosa cells from FasL-induced apoptosis by stimulating the PI3K/Akt pathway. Furthermore, the protective effect of IGF-I is dependent on unperturbed progression through the cell cycle regulated by the PI3K/Akt pathway.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
). Phosphorylation of Akt was detected at t = 0 h, before treatment with IGF-I. p-Akt appeared to increase 5 min after the addition of IGF-I, and the increase was statistically significant by 30 min. Pretreatment with the PI3K inhibitor, LY294002, inhibited phosphorylation of Akt by IGF-I, whereas the MEK (MAPK pathway) inhibitor, PD98059, had no significant effect. The two forms of p-ERK, p-ERK-1 (p44) and p-ERK-2 (p42), were detected at t = 0 h, before treatment with IGF-I. p-ERK-1/2 increased significantly 10 min after addition of IGF-I and remained elevated at 60 min. Pretreatment with PD98059 inhibited phosphorylation of ERK-1/2 by IGF-I, but treatment with LY294002 had no significant effect.
|
). Pretreatment with the PI3K inhibitor, LY294002, before the addition of IGF-I, completely blocked the protective effect of IGF-I against FasL-induced killing. Pretreatment with the MEK inhibitor, PD98059, did not alter the protective effect of IGF-I. These results indicate that the PI3K pathway, but not the MAPK pathway, is necessary for the protective effect of IGF-I against FasL-induced killing.
|
, B and C). Pretreatment with LY294002 prevented protection by IGF-I against FasL, but pretreatment with PD98059 had no effect. Pretreatment with either LY294002 or PD98059 did not affect the number of apoptotic cells in cultures not treated with FasL.
As an additional measure of apoptosis, cell lysates were assayed for caspase-3 activity. In both control cultures and cultures pretreated with IGF-I, caspase-3 activity increased significantly 1 h after treatment with FasL and was significantly higher in control vs. IGF-I-treated cultures at 2 h (Fig. 3A). Cumulative caspase-3 activity during 24 h after FasL was higher in control cells vs. cells treated with IGF-I (Fig. 3B).
|
). Pretreatment with LY294002 prevented the protective effect of IGF-I, but PD98059 had no effect. Therefore, several different assays for cell viability and apoptosis indicate that IGF-I reduces FasL-induced apoptosis through the PI3K pathway.
Activation of Akt Is Required for the Protective Effect of IGF-I Against Fas-Mediated Killing
To determine whether Akt is required for the protective effect of IGF-I, granulosa cells were infected with adenoviruses expressing constitutively active Akt (myrAkt) or dominant negative Akt (dnAkt). The myrAkt adenovirus expresses murine Akt that has a c-src myristoylation sequence and a hemagglutinin (HA) tag fused to the amino terminus of wild-type murine Akt (25). The dnAkt adenovirus encodes a murine Akt mutant in which the two major phosphorylation sites (Thr308 and Ser473) and the phosphate transfer residue in the catalytic site (Lys179) are replaced by Ala. dnAkt is constructed with HA and green fluorescence protein (GFP) tags for detection (26, 27). A null virus that was used as a control adenoviral vector contains no cDNA insert (28). Infection efficiency at 24 h was assessed by analyzing expression of GFP in cells infected with dnAkt. Approximately 90% of granulosa cells infected with 10 multiples of infection of dnAkt adenovirus expressed GFP (Fig. 4A). In subsequent experiments, all adenoviruses were used at 10 multiples of infection. At 48 h after infection, total Akt and HA were elevated in cells infected with myrAkt and dnAkt but were not detected in noninfected cells or cells infected with null adenovirus (Fig. 4B).
|
). In cells infected with myrAkt, FasL-induced killing was reduced by 59% compared with null-infected cells (P < 0.05), and pretreatment with IGF-I did not further reduce killing. In cultures infected with dnAkt, 38% more cells were killed by FasL than in null-infected cells (P < 0.05), and pretreatment with IGF-I conferred no protection from FasL-induced killing. These results show that activation of Akt is necessary for the protective effect of IGF-I against FasL-induced killing.
|
). The percent of cells in G2/M phases was not affected. Inhibition of the PI3K pathway with LY294002 decreased progression from G0/G1 to S phase: the percent of cells in G0/G1 was increased, and the percent of cells in S was decreased compared with control cultures (P < 0.05; Table 1). In the presence of LY294002, the IGF-I-mediated increase in progression from G0/G1 to S phase was blocked. Inhibition of the MAPK pathway with PD98059 resulted in no changes in cell cycle distribution in the absence of IGF-I. However, treatment with PD98059 blocked the ability of IGF-I to promote cell cycle progression (Table 1). These results show that a functional PI3K pathway is required for progression from G1 to S phase in basal media, and that both the PI3K and MAPK pathways are involved in increased cell cycle progression induced by IGF-I.
|
). In cells expressing myrAkt, IGF-I did not further increase the percent of cells in S phase. Expression of dnAkt reduced cell cycle progression vs. null-infected cells, as shown by a reduced percent of cells in S phase. In cells expressing dnAkt, the ability of IGF-I to promote cell cycle progression was abolished. Therefore, stimulation of cell cycle progression by IGF-I requires activation of Akt.
|
). Therefore, at least one mechanism for the stimulation of cell cycle progression, inhibition of p27Kip1, is regulated by Akt in granulosa cells.
Inhibition of Cell Cycle Progression Prevents the Protective Effect of IGF-I Against FasL-Induced Apoptosis
Experiments were performed to test whether cell cycle progression was required for the protective effect of IGF-I against FasL. Cells were treated with roscovitine, an inhibitor of cdk2 activity that blocks transition from G1 to S phase and G2 to M phase, or with hydroxyurea, which blocks DNA synthesis by inhibition of ribonucleotide reductase and causes cells to remain in S phase. In control cultures, treatment of granulosa cells with IGF-I decreased FasL-induced killing by 31% (Fig. 6). However, in the presence of roscovitine, IGF-I had no protective effect against killing. Cell cycle analysis confirmed that roscovitine inhibited the cell cycle as expected: in cells treated with roscovitine, the percent of cells in G0/G1 phases was increased, and the percent in S phase was 34% lower compared with control cultures (P < 0.05; Table 3). Treatment with roscovitine prevented the effect of IGF-I to increase progression from G0/G1 to S phase. These results are consistent with cell cycle blockage by roscovitine at the G1/S transition and show that IGF-I does not overcome the blockage. In cells pretreated with hydroxyurea, IGF-I decreased FasL-induced killing by 21% (P < 0.05; Fig. 6). The percent of hydroxyurea-treated cells in G0/G1 phases and in G2/M phases was less than controls, whereas the percent of cells in S phase was 54% higher (P < 0.05; Table 3), consistent with cell cycle blockage in S phase. Treatment of cells with hydroxyurea plus IGF-I resulted in even more cells in S phase (P < 0.05 vs. hydroxyurea alone), suggesting that IGF-I increased progression from G0/G1 to S phase but did not overcome the blockage in S phase by hydroxyurea. These data show that when cells were blocked at the G1/S transition, IGF-I did not provide protection from apoptosis, but that when cells were blocked in S phase, some protection by IGF-I was maintained.
|
|
). Addition of roscovitine blocked the protection by myrAkt and increased FasL-induced killing by 150% (P < 0.05) compared with myrAkt-infected cells alone. Cell cycle analysis showed that the percent of cells in S phase was increased in cells expressing myrAkt but that this effect was inhibited by roscovitine (P < 0.05; Table 4). These results suggest that the protective effect of Akt against FasL-induced apoptosis requires active cdk2 and/or cell cycle progression from G1 to S phase.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
IGF-I stimulated both the PI3K and MAPK pathways in granulosa cells, as evidenced by increased phosphorylation of Akt and ERK-1/2, respectively. Whereas the PI3K inhibitor, LY294002, prevented the protective effect of IGF-I against FasL-induced apoptosis, the MEK inhibitor, PD98059, had no effect. Therefore, the protective effect of IGF-I is mediated by the PI3K pathway and not by the MAPK pathway. These results are consistent with reports that survival of granulosa cells in serum-free culture medium in response to IGF-I is associated with increased phosphorylation of Akt (35) and increased Akt kinase activity (36). In the current study, a dominant negative form of Akt prevented the protective effect of IGF-I whereas constitutively active myrAkt mimicked the action of IGF-I and protected cells from killing by FasL. These results are the first to demonstrate that activation of Akt kinase, downstream of PI3K, is required for protection of granulosa cells by IGF-I. The results are consistent with observations in fibroblasts and neuronal cells in which IGF-I stimulated both the PI3K and MAPK pathways, but only the PI3K pathway was required for protection against apoptosis (37, 38, 39). In contrast, in some cell types, IGF-I-mediated protection from apoptosis appears to occur through both the PI3K and MAPK pathways (40, 41). Our findings in granulosa cells are similar to results obtained using endothelial cells and various cell lines in which activation of the PI3K/Akt pathway inhibited FasL-induced apoptosis (42, 43, 44). The fact that Fas-mediated apoptosis is impaired in phosphatase and tensin homologue (PTEN)-mutant mice, in which phosphorylation of Akt is abnormally elevated, also supports a role for the PI3K/Akt pathway in regulating susceptibility to FasL-induced apoptosis (45).
Previous studies in our laboratory showed that growth factors, including IGF-I, epidermal growth factor, and basic fibroblast factor, which effectively suppressed FasL-induced apoptosis, also appeared to increase proliferation of bovine granulosa cells in vitro (9). These results suggested the possibility that progression through the cell cycle alters susceptibility to apoptosis. In the current study, analysis of the distribution of granulosa cells in various stages of the cell cycle by flow cytometry showed that IGF-I increased progression from G0/G1 to S phase. Furthermore, analysis of BrdU incorporation into DNA showed that treatment with IGF-I increased synthesis of DNA. The protective effect of IGF-I against FasL-induced apoptosis was prevented when progression from G1 to S phase of the cell cycle through the PI3K/Akt pathway was perturbed. Blocking the PI3K/Akt pathway by treatment with LY294002 or expression of dnAkt inhibited G1 to S phase progression in basal media (containing 100 ng/ml insulin) and prevented the increase in G1 to S phase progression in response to IGF-I. Expression of constitutively active myrAkt increased G1 to S phase progression. These results indicate that the PI3K/Akt pathway regulates cell cycle progression as well as survival. The fact that treatment with the cdk2 inhibitor, roscovitine, inhibited G1 to S phase progression and prevented the protective effects of IGF-I and myrAkt against FasL-induced apoptosis indicates that cell cycle progression through the PI3K/Akt pathway is necessary for protection from apoptosis. Treatment with the MAPK pathway inhibitor, PD98059, prevented the increase in G1 to S phase progression in response to IGF-I but did not alter G1 to S phase progression in basal media or the extent of FasL-induced killing. These results indicate that stimulation of the MAPK pathway by IGF-I and the associated increase in G1 to S phase progression is not sufficient for protection against apoptosis.
A number of substrates of activated Akt that regulate cell survival and proliferation have been identified. Proteins that are phosphorylated and inactivated by Akt include the apoptosis-inducing protease, caspase-9, and the proapoptotic Bcl-2 family member, Bad (46, 47). Akt also phosphorylates the transcription factor, forkhead, causing it to be localized in the cytoplasm. In the absence of active Akt, nonphosphorylated forkhead localizes in the nucleus where it can either induce transcription of FasL and thereby promote apoptosis, or stimulate transcription of p27Kip1 and induce withdrawal from the cell cycle (31, 48, 49). Another mechanism by which Akt is thought to prevent apoptosis is by activating the transcription factor nuclear factor-
B (NFB), which in turn induces the expression of antiapoptotic genes (50, 51). Furthermore, Akt stimulates the expression of the antiapoptotic protein, FADD-like ICE (FLICE)-inhibitory protein (FLIP), which inhibits activation of caspase-8 (42, 52). Akt is thought to modulate the cell cycle through its effects on several cell cycle-regulatory proteins. Akt inactivates forkhead transcription factors and thus prevents transcriptional activation of the cdk inhibitor p27Kip1 (31, 49). Akt also phosphorylates and inactivates glycogen synthase kinase-3ß (GSK3). Active glycogen synthase kinase-3ß phosphorylates cyclin D1, leading to its degradation (53). Akt thus promotes cell cycle progression by increasing levels of cyclin D1 and decreasing levels of p27Kip1. In the current study, expression of dnAkt increased levels of p27Kip1, suppressed G1 to S phase progression, and prevented the effect of IGF-I to protect against FasL-induced apoptosis. Our results demonstrate that activation of Akt in granulosa cells is a key step in a pathway that regulates cell cycle progression as well as survival.
There is evidence for a relationship between stage of the cell cycle and the susceptibility to apoptosis (20, 21, 22, 23). In general, apoptosis is detected in vivo at the highest frequency in proliferating rather than quiescent tissues. Certain cell types appear to be most susceptible to undergoing apoptosis during the transition from G1 to S phase (20). For example, in lymphocytes undergoing activation-induced cell death, a process mediated by Fas/FasL interactions, cells undergo apoptosis at the G1 to S transition (54). In contrast, cells that are arrested in early G1 or G0 of the cell cycle may be resistant to apoptosis. This includes cell types such as myocytes, in which withdrawal from the cell cycle upon terminal differentiation is associated with resistance to apoptosis (55). Furthermore, treatment of T cells with agents that block the cell cycle in early G1 induces resistance to apoptosis, whereas blocking at the G1/S transition induces sensitivity to apoptosis (20). The Bcl-2 protein family, which is well recognized for its control over apoptosis, also modulates the cell cycle. Overexpression of Bcl-2 protects against apoptosis and also prevents quiescent cells from entering the cell cycle (56, 57). The results of the current study provide further evidence for a relationship between the cell cycle and susceptibility to apoptosis.
The ability of IGF-I to prevent Fas-mediated apoptosis may play a critical role in promoting follicle development. Expressions of Fas and FasL are low in healthy follicles and are elevated in atretic follicles in cows, rodents, and humans (4, 5, 6, 7, 8). Granulosa cells from atretic bovine follicles are more susceptible to FasL-induced apoptosis in vitro than cells from healthy follicles (4). These findings indicate that the Fas pathway becomes activated in follicles undergoing atresia but is suppressed in healthy follicles. It is likely that the greater bioavailability of IGF-I within healthy follicles, compared with atretic follicles (16), contributes to inhibition of the Fas-mediated death pathway. Support for a role of IGF-I in suppressing Fas-mediated apoptosis has been provided by experiments with cultured bovine granulosa cells. Withdrawal of serum from granulosa cell cultures stimulates expression of Fas and FasL and induces apoptosis through Fas/FasL interaction (10). IGF-I was identified as at least one of the factors present in serum that prevents FasL-induced apoptosis of bovine granulosa cells (9). The ability of locally produced growth factors such as IGF-I to suppress expression of Fas and FasL, and to render granulosa cells resistant to Fas-mediated apoptosis, is likely to be important for promotion of follicle development and survival.
In summary, the effect of IGF-I to protect granulosa cells from apoptosis is mediated through the PI3K/Akt pathway and requires that PI3K/Akt-mediated progression from G1 to S phase of the cell cycle is not perturbed. Furthermore, the ability of activated Akt to protect cells against apoptosis is prevented when cell cycle progression is blocked. These results suggest that effects of activated Akt on substrates known to be involved in antiapoptotic pathways (such as Bad and forkhead transcription factors) are not adequate to protect cells against apoptosis when cell cycle progression is prevented. An interdependency between cell proliferation and survival is relevant to the physiology of ovarian follicle development. Granulosa cells are dependent upon growth factors for survival during the period of proliferation in the developing follicle. Stimulation of the PI3K/Akt pathway by IGF-I may promote cell survival by suppression of apoptotic pathways and by concomitantly maintaining appropriate progression through the cell cycle.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cell Isolation, Preparation, and Culture
Freshly excised cow ovaries were obtained from an abattoir, transported in saline at 4 C (
1.5 h), and processed immediately. Granulosa cells were collected by aspiration of 2- to 4-mm follicles and cultured in DMEM-F12 (supplemented with 1 mM pyruvate, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone) containing 10% fetal bovine serum. Cells were plated (d 0) at 1 x 105 cells per well in 96-well plates for cell viability assays, at 1.5–2 x 106 cells per well in 35-mm dishes for immunoblotting and flow cytometric analyses, and at 3 x 105 cells per well in 24-well plates for assays of caspase-3 activity. In all experiments except those involving adenoviral infection (described below), media were changed on d 1 to serum-free DMEM-F12 supplemented as above and containing 100 ng/ml insulin, 5 µg/ml transferrin, 20 nM Na-selenite and 0.1% BSA (ITS). The concentration of insulin in ITS was experimentally determined to be the minimum required to prevent the cells from undergoing spontaneous apoptosis (data not shown). Insulin concentrations in commercial preparations of ITS are much higher. On d 2, cells were treated with 0 or 100 ng/ml IGF-I (t = 0 h) and, where appropriate, with 0 or 100 ng/ml FasL at t = 4 h. In some experiments, parallel cultures were pretreated 0.5 h before IGF-I with specific second messenger or cell cycle inhibitors. The inhibitors used were: LY294002 (20 µM), a PI3K inhibitor; PD98059 (30 µM), a MEK (MAPK kinase) inhibitor; roscovitine (20 µM), a cdk2 inhibitor which blocks cells at the G1/S interface; and hydroxyurea (25 µg/ml), a ribonucleotide reductase inhibitor that blocks cells in S phase. Cell viability assays and flow cytometric analyses were performed 24 h after treatment. Cells were collected for immunoblotting or assay of caspase-3 activity at various times from 0–24 h after treatment.
Expression of Recombinant Akt Proteins by Adenoviral Infection
On d 2 after plating, bovine granulosa cells at approximately 90% confluency were infected with recombinant adenoviruses (myrAkt, dnAkt, and null) in DMEM-F12 containing 10% fetal bovine serum. On d 3, media were changed to DMEM-F12-ITS, and various treatments were applied. On d 4, cell viability assays were performed and cells were collected for immunoblotting or cell cycle analysis.
Assays for Cell Viability and Activated Caspase-3
The number of viable cells attached to each culture dish at the end of the culture period was determined. Cells were trypsinized, collected, and stained with trypan blue, and live cells were counted in a hemacytometer. The percent of granulosa cells killed by FasL was calculated by comparing the number of live cells present in FasL-treated cultures vs. the number present in control cultures that received the same pretreatment without addition of FasL. Treatments within each experiment were done in quadruplicate wells of 96-well culture plates. This assay provides the best quantitative measure of the percentage of granulosa cells susceptible to FasL-induced apoptosis. Assays for caspase-3 activity and cellular DNA content (described below) were used to confirm that cell death occurred by apoptosis.
Caspase-3 activity in cell lysates was determined by measuring the amount of fluorescent-labeled caspase-3-specific substrate (R110-DEVD) cleaved per hour using the EnzChek caspase-3 assay kit (Molecular Probes, Eugene, OR). Activity is expressed as picomoles substrate cleaved/µg protein per h.
Levels of immunoreactive active caspase-3 were determined by flow cytometric analysis of cells obtained 12 h after treatment with 0 or 100 ng/ml FasL. Cells were trypsinized, collected, and fixed in 80% ethanol. Cells were incubated with 1 µg/ml antiactive caspase-3 antibody in PBS containing 1% calf serum, 0.1% saponin, and 0.1% sodium azide at room temperature for 2 h. Cells were rinsed, and 0.5 µg/ml Alexa 488-conjugated goat antirabbit IgG was used to detect the primary antibody. Log fluorescence of Alexa 488 was detected in 10,000 cells on a FACScan flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ). Events were gated for single cells based on side scatter vs. forward scatter, and the mean fluorescent intensity of single cells was determined for each sample.
Immunoblotting
Cells from duplicate wells were scraped into ice-cold HEPES-buffered saline (10 mM HEPES, 150 mM NaCl), pelleted, and suspended in RIPA buffer (1.0% Nonidet P-40, 0.05% Na-deoxycholate, 0.1% SDS in PBS) containing freshly added proteinase inhibitors (100 µg/ml phenylmethylsulfonyl fluoride and 3 mg/ml aprotinin). Cells were sonicated for 10 sec, and lysates were frozen until further analysis. Protein content of cell lysates was determined using the DC Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).
Protein lysates (60–120 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in Tris-buffered saline (TBST; 20 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween-20) containing 5% nonfat milk for 30 min at room temperature. Membranes were incubated at 4 C overnight in TBST-5% BSA containing antibodies to p-Akt, p-ERK, or HA (dilution 1:1000) or p27Kip1 (dilution 1:500). Membranes were washed, incubated with horseradish peroxidase-conjugated secondary antibodies in TBST-5% nonfat milk for 30 min at room temperature, and washed. A chemiluminescent signal was generated using Western Blot Chemiluminescence Reagent (NEN Life Science Products, Boston, MA), and membranes were exposed to x-ray film (Eastman Kodak, Rochester, NY). After detection of p-Akt, p-ERK, and p27Kip1, membranes were stripped and then reprobed with antibodies for total Akt or ERK-2 (dilution 1:1000) or ß-actin (dilution 1:5000), respectively. Signals were quantified by densitometry of digitized images using Scion Image Software (Scion Corp., Frederick, MD), and ratios of p-kinases to total kinases and p27Kip1 to ß-actin were calculated.
Measurement of Proliferation by BrdU Incorporation and Detection
Granulosa cells in DMEM-F12-ITS were treated on d 2 of culture with 0 or 10 µM BrdU and 0 or 100 ng/ml IGF-I. After 24 h, cells were trypsinized, resuspended in DMEM-F12, and fixed in 80% ethanol. Detection of BrdU was performed as previously described with minor modifications (58). Briefly, cells were pretreated with 100 µg/ml RNase A for 20 min, rinsed in PBS, and treated with 0.1 M Na-citrate in 0.5% Triton X-100 in PBS for 10 min on ice. Cells were rinsed in 0.01 M Tris buffer containing 10 mM MgCl2, DNA was partially digested by addition of 30 U/ml BamHI for 30 min, and cells were rinsed in Tris buffer. Cells were stained with 2 µg/ml anti-BrdU antibody in PBS containing 0.5% BSA and 0.5% Tween 20 at room temperature for 2 h and rinsed, and 0.5 µg/ml Alexa 488-conjugated goat antimouse IgG was used to detect the BrdU antibody. The cells were counterstained with PI to measure the DNA content, and 20,000 cells were analyzed for both BrdU and PI fluorescence on a FACScan flow cytometer (Becton Dickinson and Co.). Events were gated for single cells based on PI fluorescence. Within each experimental replicate, a fluorescent threshold for identification of positive cells was established based on negative control cells that received no BrdU and were processed as described above. The threshold was chosen such that >95% of the cells that did not receive BrdU were negative.
Flow Cytometric Determination of Cellular DNA Content: Analysis of Apoptosis and the Cell Cycle
Cellular DNA content was determined by flow cytometric measurement of PI binding. Adherent granulosa cells in six-well plates were trypsinized, resuspended in DMEM-F12, and fixed in 80% ethanol. Cells were stained with 5 µg/ml PI in 0.01 M PBS containing 0.01% Triton X-100 and 30 µg/ml DNase-free RNase A. Cells (10,000 per sample) were analyzed by flow cytometry. Data were gated for single cells. In experiments to determine the distribution of cells in various stages of the cell cycle, DNA content was assigned to G0/G1, S or G2M phases based on the method of Ormerod (59) using WinMDI software (The Scripps Research Institute, La Jolla, CA). Cell cycle data presented in Tables 1 and 3 were performed and statistically analyzed as a single experiment but are presented separately for clarity. In experiments to measure the effects of various treatments on FasL-induced apoptosis, the percentage of cells with less than diploid content of DNA (A0 peak) was determined.
Statistical Analysis
All experiments were repeated using at least three separate preparations of granulosa cells. Caspase-3 activity in cell lysates and Akt and ERK immunoblotting densities were analyzed by two-way ANOVA using a randomized complete block design. Linear contrast was performed to compare individual means when overall significance was observed (60). All other data were analyzed by one-way ANOVA using a randomized complete block design, and Duncan’s new multiple range test was used for comparison of means when overall significance was observed (61).
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Abbreviations: BrdU, Bromodeoxyuridine; cdk, cyclin-dependent kinase; dnAkt, dominant negative Akt; FasL, Fas ligand; GFP, green fluorescent protein; HA, hemagglutinin; ITS, 100 ng/ml insulin, 5 µg/ml transferrin, 20 nM Na-selenite and 0.1% BSA; MEK, MAPK kinase; myrAkt, constitutively active Akt; p-Akt, phosphorylated Akt; p-ERK, phosphorylated ERK; PI, propidium iodide; PI3K, phosphoinositide 3'-OH kinase.
Received for publication May 14, 2003. Accepted for publication October 23, 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
B is a target of Akt in anti-apoptotic PDGF signaling. Nature 401:86–90[CrossRef][Medline]
B activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401:82–85[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  L. Tosca, C. Rame, C. Chabrolle, S. Tesseraud, and J. Dupont Metformin decreases IGF1-induced cell proliferation and protein synthesis through AMP-activated protein kinase in cultured bovine granulosa cells Reproduction, February 1, 2010; 139(2): 409 - 418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-M. Chang, W.-H. Kuo, T.-Y. Lai, Y.-T. Shih, F.-J. Tsai, C.-H. Tsai, W.-T. Shu, Y.-Y. Chen, Y.-S. Chen, W.-W. Kuo, et al. RSC96 Schwann Cell Proliferation and Survival Induced by Dilong through PI3K/Akt Signaling Mediated by IGF-I Evid. Based Complement. Altern. Med., December 29, 2009; (2009) nep216v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Iwase, M. Goto, T. Harata, S. Takigawa, T. Nakahara, K. Suzuki, S. Manabe, and F. Kikkawa Insulin Attenuates the Insulin-Like Growth Factor-I (IGF-I)-Akt Pathway, not IGF-I-Extracellularly Regulated Kinase Pathway, in Luteinized Granulosa Cells with an Increase in PTEN J. Clin. Endocrinol. Metab., June 1, 2009; 94(6): 2184 - 2191. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zheng, D. Boerboom, and P. D Carriere Transforming growth factor-{beta}1 inhibits luteinization and promotes apoptosis in bovine granulosa cells Reproduction, June 1, 2009; 137(6): 969 - 977. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Cannon, S. V. Seekallu, C. A. VandeVoort, and C. L. Chaffin Association of luteinizing hormone receptor gene expression with cell cycle progression in granulosa cells Am J Physiol Endocrinol Metab, June 1, 2009; 296(6): E1392 - E1399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Goto, A. Iwase, T. Harata, S. Takigawa, K. Suzuki, S. Manabe, and F. Kikkawa IGF1-induced AKT phosphorylation and cell proliferation are suppressed with the increase in PTEN during luteinization in human granulosa cells Reproduction, May 1, 2009; 137(5): 835 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M A Velazquez, J Zaraza, A Oropeza, R Webb, and H Niemann The role of IGF1 in the in vivo production of bovine embryos from superovulated donors Reproduction, February 1, 2009; 137(2): 161 - 180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Ben-Ami, L. Armon, S. Freimann, D. Strassburger, R. Ron-El, and A. Amsterdam EGF-like growth factors as LH mediators in the human corpus luteum Hum. Reprod., January 1, 2009; 24(1): 176 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sen, L. Lv, N. Bello, J. J. Ireland, and G. W. Smith Cocaine- and Amphetamine-Regulated Transcript Accelerates Termination of Follicle-Stimulating Hormone-Induced Extracellularly Regulated Kinase 1/2 and Akt Activation by Regulating the Expression and Degradation of Specific Mitogen-Activated Protein Kinase Phosphatases in Bovine Granulosa Cells Mol. Endocrinol., December 1, 2008; 22(12): 2655 - 2676. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Segrelles, J. Lu, B. Hammann, M. Santos, M. Moral, J. L. Cascallana, M. F. Lara, O. Rho, S. Carbajal, J. Traag, et al. Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis Cancer Res., November 15, 2007; 67(22): 10879 - 10888. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, V. Chandrakanthan, M. L Day, and C. O'Neill Direct Evidence for the Action of Phosphatidylinositol (3,4,5)-Trisphosphate-Mediated Signal Transduction in the 2-Cell Mouse Embryo Biol Reprod, November 1, 2007; 77(5): 813 - 821. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sen, A. Bettegowda, F. Jimenez-Krassel, J. J. Ireland, and G. W. Smith Cocaine- and Amphetamine-Regulated Transcript Regulation of Follicle-Stimulating Hormone Signal Transduction in Bovine Granulosa Cells Endocrinology, September 1, 2007; 148(9): 4400 - 4410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Neganova, H. Al-Qassab, H. Heffron, C. Selman, A. I. Choudhury, S. J. Lingard, I. Diakonov, M. Patterson, M. Ghatei, S. R. Bloom, et al. Role of Central Nervous System and Ovarian Insulin Receptor Substrate 2 Signaling in Female Reproductive Function in the Mouse Biol Reprod, June 1, 2007; 76(6): 1045 - 1053. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K E Ryan, S M Casey, M J Canty, M A Crowe, F Martin, and A C O Evans Akt and Erk signal transduction pathways are early markers of differentiation in dominant and subordinate ovarian follicles in cattle Reproduction, March 1, 2007; 133(3): 617 - 626. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Orisaka, S. Orisaka, J.-Y. Jiang, J. Craig, Y. Wang, F. Kotsuji, and B. K. Tsang Growth Differentiation Factor 9 Is Antiapoptotic during Follicular Development from Preantral to Early Antral Stage Mol. Endocrinol., October 1, 2006; 20(10): 2456 - 2468. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Gervais, C. Dugourd, L. Muller, C. Ardidie, B. Canton, L. Loviconi, P. Corvol, H. Chneiweiss, and C. Monnot Akt Down-Regulates ERK1/2 Nuclear Localization and Angiotensin II-induced Cell Proliferation through PEA-15 Mol. Biol. Cell, September 1, 2006; 17(9): 3940 - 3951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M Quirk, R. G Cowan, and R. M Harman The susceptibility of granulosa cells to apoptosis is influenced by oestradiol and the cell cycle. J. Endocrinol., June 1, 2006; 189(3): 441 - 453. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A Slot, J. Kastelijn, A. Bachelot, P. A Kelly, N. Binart, and K. J Teerds Reduced recruitment and survival of primordial and growing follicles in GH receptor-deficient mice. Reproduction, March 1, 2006; 131(3): 525 - 532. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A Slot, M. Voorendt, M. de Boer-Brouwer, H. H van Vugt, and K. J Teerds Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary J. Endocrinol., February 1, 2006; 188(2): 179 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Park, E. T. Maizels, Z. J. Feiger, H. Alam, C. A. Peters, T. K. Woodruff, T. G. Unterman, E. J. Lee, J. L. Jameson, and M. Hunzicker-Dunn Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad J. Biol. Chem., March 11, 2005; 280(10): 9135 - 9148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Quirk, R. G. Cowan, and R. M. Harman Progesterone Receptor and the Cell Cycle Modulate Apoptosis in Granulosa Cells Endocrinology, November 1, 2004; 145(11): 5033 - 5043. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Quirk, R. G. Cowan, R. M. Harman, C.-L. Hu, and D. A. Porter Ovarian follicular growth and atresia: The relationship between cell proliferation and survival J Anim Sci, January 1, 2004; 82(13_suppl): E40 - 52. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, P < 0.05 vs. other treatments at the same time point.
