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Cited by 126Reproductive Biology and FertilityFOXO transcription factor regulationOvarian function and disorders

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Involvement of FoxO1 in the effects of follicle-stimulating hormone on inhibition of apoptosis in mouse granulosa cells

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Shen, M, Liu, Z, Li, B, Teng, Y, Zhang, J, Tang, Y, Sun, S-C, Liu, H·2014·Cell Death & Disease
DOI 10.1038/cddis.2014.400PMC PMC4237239
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Abstract

In mammalian ovaries, follicular atresia occurs periodically and destroys almost all the follicles in the ovary. Follicle-stimulating hormone (FSH) acts as the primary survival factor during follicular atresia by preventing apoptosis in granulosa cells. FoxO1 is a critical factor in promoting follicular atresia and granulosa cell apoptosis. FSH inhibits the induction of FoxO1. In this report, we investigated the role of FSH-FoxO1 pathway in mouse follicular atresia. FSH dampened stress-induced apoptosis and the expression of FoxO1 and pro-apoptosis genes in mouse granulosa cells (MGCs). In contrast, overexpression of FoxO1 inhibited the viability of MGCs and induced the expression of endogenous FoxO1. The signaling cascades involved in regulating FoxO1 activity upon FSH treatment were identified using FSH signaling antagonists. Blocking protein kinase A (PKA), phosphatidylinositol-3 kinase (PI3K) or protein kinase B (AKT) restored the upregulation of FoxO1 and apoptotic signals, which was suppressed by FSH. Moreover, inhibition of PKA or PI3K impaired FSH-induced AKT activity, but inactivation of PI3K or AKT had little effect on PKA activity in the presence of FSH. Correspondingly, constitutive activation of FoxO1 (all three AKT sites were replaced by alanines) also promoted MGC apoptosis despite FSH administration. Furthermore, both luciferase reporter assays and chromatin immunoprecipitation assays showed that FoxO1 directly bound to a FoxO-recognized element site within the FoxO1 promoter and contributed to the regulation of FoxO1 expression in response to FSH. Taken together, we propose a novel model in which FSH downregulates FoxO1-dependent apoptosis in MGCs by coordinating the PKA–PI3K–AKT–FoxO1 axis and FoxO1–FoxO1 positive feedback.

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Figure 1 FSH reduced apoptosis in MGCs of ovarian DFs. Mice were injected i.p. with FSH twice daily (12-h intervals, 08 00 and 2000 hours) for 2 days at a dose of 10 IU on day 1 and 5 IU on day 2. FSH was then withdrawn for an additional 24 or 48 h, or injected i.p. (10 IU per mouse) 6 h before MGC collection. (a–e) At 48, 72 and 96 h after the first FSH injection, the right ovaries were paraffin-embedded and serially sectioned for the TUNEL assay (a) and H&E staining (b). MGCs were isolated from DFs (>200 μm) in the left ovaries for caspase activity detection (c–e). The data represent the mean±S.E. (n=3). *P<0.05, Student's t-test. Bar, 100 μm. O, oocyte; GC, granulosa cells; CL, corpus luteum
Figure 1 FSH reduced apoptosis in MGCs of ovarian DFs. Mice were injected i.p. with FSH twice daily (12-h intervals, 08 00 and 2000 hours) for 2 days at a dose of 10 IU on day 1 and 5 IU on day 2. FSH was then withdrawn for an additional 24 or 48 h, or injected i.p. (10 IU per mouse) 6 h before MGC collection. (a–e) At 48, 72 and 96 h after the first FSH injection, the right ovaries were paraffin-embedded and serially sectioned for the TUNEL assay (a) and H&E staining (b). MGCs were isolated from DFs (>200 μm) in the left ovaries for caspase activity detection (c–e). The data represent the mean±S.E. (n=3). *P<0.05, Student's t-test. Bar, 100 μm. O, oocyte; GC, granulosa cells; CL, corpus luteumCC BY · PubMed Central
Figure 2 FSH protected follicular MGCs from apoptosis in a FoxO1-dependent manner. Ovarian stimulation and MGC retrieval were performed as described in Supplementary Figure S1. (a) The expression of FoxO family members in MGCs of DFs was measured with qRT-PCR. (b) The responsiveness of FoxO1 and FoxO3a to FSH treatment was detected as above. (c) Transcription changes in FoxO1 in response to FSH-induced MGC protection. (d) The inhibition of FoxO1 expression by FSH was examined using western blotting. α-Tubulin served as the loading control. (e) Quantification of relative FoxO1 protein levels with densitometry. ImageJ 1.42q software was used to analyze the density of each band in Figure 2d, and the relative expression level was normalized to that of α-tubulin. The data represent the mean±S.E. (n=3). *P<0.05; **P<0.01, Student's t-test
Figure 2 FSH protected follicular MGCs from apoptosis in a FoxO1-dependent manner. Ovarian stimulation and MGC retrieval were performed as described in Supplementary Figure S1. (a) The expression of FoxO family members in MGCs of DFs was measured with qRT-PCR. (b) The responsiveness of FoxO1 and FoxO3a to FSH treatment was detected as above. (c) Transcription changes in FoxO1 in response to FSH-induced MGC protection. (d) The inhibition of FoxO1 expression by FSH was examined using western blotting. α-Tubulin served as the loading control. (e) Quantification of relative FoxO1 protein levels with densitometry. ImageJ 1.42q software was used to analyze the density of each band in Figure 2d, and the relative expression level was normalized to that of α-tubulin. The data represent the mean±S.E. (n=3). *P<0.05; **P<0.01, Student's t-testCC BY · PubMed Central
Figure 3 FSH suppressed apoptosis in cultured MGCs. (a) Granulosa cells collected from DFs were cultured in serum-free medium containing 200 μM H2O2 and FSH at different concentrations for 6 h. The apoptotic signals were visualized with TUNEL staining (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (b) Quantification of the apoptosis rates by calculating the average number of TUNEL-positive nuclei per visual field. Experiments were repeated three times, and cells were counted in three randomly selected fields from each coverslip. Data represent the means±S.E. The statistical significance between groups was analyzed with one-way ANOVA, and bars labeled with different letters differ significantly at P<0.01 (a and b) or P<0.05 (a and b). (c) After incubation with 200 μM H2O2 for 0 (vehicle), 1, 2 or 3 h, MGCs were rinsed with PBS and cultured in serum-free medium containing 7.5 IU/ml FSH for 12 h. Apoptosis was detected as above. Bar, 20 μm. (d) Quantification of apoptosis rates in each treatment as shown in (c). Data represent the means±S.E. (e–g) MGCs were incubated for 1 h in 200 μM H2O2, washed with PBS, plated in serum-free medium with 7.5 IU/ml FSH for 12 h, and then processed for qRT-PCR for Bim (e), caspase-3 activity assay (f) and intracellular ROS detection (g). Bar, 50 μm. (h) Quantification of intracellular ROS levels. The optical density was evaluated in each MGC with ImageJ 1.42q software. Data represent mean±S.E. (n=3). *P<0.05; **P<0.01, Student's t-test
Figure 3 FSH suppressed apoptosis in cultured MGCs. (a) Granulosa cells collected from DFs were cultured in serum-free medium containing 200 μM H2O2 and FSH at different concentrations for 6 h. The apoptotic signals were visualized with TUNEL staining (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (b) Quantification of the apoptosis rates by calculating the average number of TUNEL-positive nuclei per visual field. Experiments were repeated three times, and cells were counted in three randomly selected fields from each coverslip. Data represent the means±S.E. The statistical significance between groups was analyzed with one-way ANOVA, and bars labeled with different letters differ significantly at P<0.01 (a and b) or P<0.05 (a and b). (c) After incubation with 200 μM H2O2 for 0 (vehicle), 1, 2 or 3 h, MGCs were rinsed with PBS and cultured in serum-free medium containing 7.5 IU/ml FSH for 12 h. Apoptosis was detected as above. Bar, 20 μm. (d) Quantification of apoptosis rates in each treatment as shown in (c). Data represent the means±S.E. (e–g) MGCs were incubated for 1 h in 200 μM H2O2, washed with PBS, plated in serum-free medium with 7.5 IU/ml FSH for 12 h, and then processed for qRT-PCR for Bim (e), caspase-3 activity assay (f) and intracellular ROS detection (g). Bar, 50 μm. (h) Quantification of intracellular ROS levels. The optical density was evaluated in each MGC with ImageJ 1.42q software. Data represent mean±S.E. (n=3). *P<0.05; **P<0.01, Student's t-testCC BY · PubMed Central
Figure 4 FoxO1 expression was downregulated by FSH in vitro. Primary MGCs collected from DFs were exposed to 200 μM H2O2 for 1 h, rinsed with PBS and cultured in serum-free medium containing 7.5 IU/ml FSH for 12 h. (a) The effects of FSH on FoxO1 expression in MGCs were determined with qRT-PCR. Expression data were normalized to that of β-actin. (b) Western blotting analysis of FoxO1 protein levels in cultured MGCs treated with or without FSH. α-Tubulin was used as the loading control. (c) Quantification of the relative FoxO1 protein level with densitometry. ImageJ 1.42q software was used to analyze the density of each band in Figure 4b, and the relative expression level was normalized to that of α-tubulin. The data represent the mean±S.E. (n=3). *P<0.05; **P<0.01, Student's t-test
Figure 4 FoxO1 expression was downregulated by FSH in vitro. Primary MGCs collected from DFs were exposed to 200 μM H2O2 for 1 h, rinsed with PBS and cultured in serum-free medium containing 7.5 IU/ml FSH for 12 h. (a) The effects of FSH on FoxO1 expression in MGCs were determined with qRT-PCR. Expression data were normalized to that of β-actin. (b) Western blotting analysis of FoxO1 protein levels in cultured MGCs treated with or without FSH. α-Tubulin was used as the loading control. (c) Quantification of the relative FoxO1 protein level with densitometry. ImageJ 1.42q software was used to analyze the density of each band in Figure 4b, and the relative expression level was normalized to that of α-tubulin. The data represent the mean±S.E. (n=3). *P<0.05; **P<0.01, Student's t-testCC BY · PubMed Central
Figure 5 FSH prevented MGCs from undergoing apoptosis via the PKA–PI3K–AKT–FoxO1 axis. Primary cultured MGCs were exposed to 200 μM H2O2 for 1 h and then grown in serum-free medium containing 7.5 IU/ml FSH for 2 h (in e, f, h and i) or 6 h (in a–d and g). H89 (10 μM), LY294002 (20 μM), SB203580 (20 μM) or U0126 (3 μM) was added for 30 min before FSH treatment. (a–d) The effects of the FSH signaling antagonists on FoxO1 expression were determined with qRT-PCR. Expression data were normalized to that of β-actin. (e) Subcellular localization of FoxO1 in response to the indicated FSH signaling inhibitors was detected using anti-FoxO1 (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (f) The percentage of cells with FoxO1 in the nucleus (orange bars) and in the cytosol (green bars). Experiments were performed in triplicate, and cells were counted in three randomly selected fields in each coverslip. Data represent the mean±S.E. (g) The effects of the FSH signaling antagonists on stress-induced apoptosis in cultured MGCs. The apoptotic signals were visualized with TUNEL staining (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (h) The effects of the PKA inhibitor H89 on the activity of PKA, PI3K and AKT. (i) The effects of the PI3K inhibitor LY294002 on the activity of PKA, PI3K and AKT. The data represent the mean±S.E. (n=3). *P<0.05; **P<0.01; ***P<0.001, Student's t-test
Figure 5 FSH prevented MGCs from undergoing apoptosis via the PKA–PI3K–AKT–FoxO1 axis. Primary cultured MGCs were exposed to 200 μM H2O2 for 1 h and then grown in serum-free medium containing 7.5 IU/ml FSH for 2 h (in e, f, h and i) or 6 h (in a–d and g). H89 (10 μM), LY294002 (20 μM), SB203580 (20 μM) or U0126 (3 μM) was added for 30 min before FSH treatment. (a–d) The effects of the FSH signaling antagonists on FoxO1 expression were determined with qRT-PCR. Expression data were normalized to that of β-actin. (e) Subcellular localization of FoxO1 in response to the indicated FSH signaling inhibitors was detected using anti-FoxO1 (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (f) The percentage of cells with FoxO1 in the nucleus (orange bars) and in the cytosol (green bars). Experiments were performed in triplicate, and cells were counted in three randomly selected fields in each coverslip. Data represent the mean±S.E. (g) The effects of the FSH signaling antagonists on stress-induced apoptosis in cultured MGCs. The apoptotic signals were visualized with TUNEL staining (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (h) The effects of the PKA inhibitor H89 on the activity of PKA, PI3K and AKT. (i) The effects of the PI3K inhibitor LY294002 on the activity of PKA, PI3K and AKT. The data represent the mean±S.E. (n=3). *P<0.05; **P<0.01; ***P<0.001, Student's t-testCC BY · PubMed Central
Figure 6 Activation of FoxO1 in MGCs abolished the anti-apoptotic effects of FSH. Primary MGCs were transfected with WT FoxO1 (FoxO1-W), a constitutively active FoxO1 mutant (FoxO1-AAA, all three AKT sites were replaced by alanines) or a control vector (pcDNA3) for 36 h. (a) Western blotting analysis of FoxO1 protein levels in cultured MGCs transfected with FoxO1 expression vectors. α-Tubulin was used as the loading control. (b) Quantification of relative FoxO1 protein levels with densitometry. ImageJ 1.42q software was used to analyze the density of each band, and the relative expression level was normalized to that of α-tubulin. (c) Cells were treated with or without FSH (7.5 IU/ml) during transfection. The apoptotic signals were detected with FITC fluorescence (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (d) Quantification of the apoptotic signals. The optical density was evaluated in each MGC with ImageJ 1.42q software. (e) Cells were incubated with or without FSH (7.5 IU/ml) before collection. Subcellular localization of FoxO1 upon FSH stimulation was visualized using anti-FoxO1 (green), and the nuclei were counterstained with DAPI (blue). Bar, 10 μm. (f) The percentage of cells with FoxO1 in the nucleus or the cytosol under the indicated conditions. Experiments were performed in triplicate, and cells were counted in three randomly selected fields on each coverslip. *P<0.05; ***P<0.001, Student's t-test
Figure 6 Activation of FoxO1 in MGCs abolished the anti-apoptotic effects of FSH. Primary MGCs were transfected with WT FoxO1 (FoxO1-W), a constitutively active FoxO1 mutant (FoxO1-AAA, all three AKT sites were replaced by alanines) or a control vector (pcDNA3) for 36 h. (a) Western blotting analysis of FoxO1 protein levels in cultured MGCs transfected with FoxO1 expression vectors. α-Tubulin was used as the loading control. (b) Quantification of relative FoxO1 protein levels with densitometry. ImageJ 1.42q software was used to analyze the density of each band, and the relative expression level was normalized to that of α-tubulin. (c) Cells were treated with or without FSH (7.5 IU/ml) during transfection. The apoptotic signals were detected with FITC fluorescence (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (d) Quantification of the apoptotic signals. The optical density was evaluated in each MGC with ImageJ 1.42q software. (e) Cells were incubated with or without FSH (7.5 IU/ml) before collection. Subcellular localization of FoxO1 upon FSH stimulation was visualized using anti-FoxO1 (green), and the nuclei were counterstained with DAPI (blue). Bar, 10 μm. (f) The percentage of cells with FoxO1 in the nucleus or the cytosol under the indicated conditions. Experiments were performed in triplicate, and cells were counted in three randomly selected fields on each coverslip. *P<0.05; ***P<0.001, Student's t-testCC BY · PubMed Central
Figure 7 FoxO1-induced apoptosis in MGCs by activating the expression of FoxO1 and downstream apoptotic genes. Primary cultured MGCs harvested from DFs were transfected with pcDNA3-FLAG-FKHR (FoxO1-W), pcDNA3-FLAG-FKHR;H215R (FoxO1-F, containing a point mutation in the DBD), or a control vector (pcDNA3) for 36 h. (a) Western blotting analysis of FoxO1 protein levels. α-Tubulin was used as the loading control. (b) Quantification of relative FoxO1 protein levels with densitometry. ImageJ 1.42q software was used to analyze the density of each band, and the relative expression level was normalized to that of α-tubulin. Yellow bar, total FoxO1 protein. Green bar, endogenous FoxO1 protein. Orange bar, exogenous FoxO1 protein. (c) The apoptotic signals in MGCs after transfection were detected with FITC fluorescence (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (d) Quantification of the apoptotic signals. The optical density was evaluated in each MGC with ImageJ 1.42q software. Experiments were performed in triplicate, and cells were counted in three randomly selected fields in each coverslip. Data represent the means±S.E. (e) Transcription changes in Bim in response to FoxO1 overexpression-induced apoptosis in MGCs. Expression data were normalized to that of β-actin. *P<0.05; **P<0.01, Student's t-test
Figure 7 FoxO1-induced apoptosis in MGCs by activating the expression of FoxO1 and downstream apoptotic genes. Primary cultured MGCs harvested from DFs were transfected with pcDNA3-FLAG-FKHR (FoxO1-W), pcDNA3-FLAG-FKHR;H215R (FoxO1-F, containing a point mutation in the DBD), or a control vector (pcDNA3) for 36 h. (a) Western blotting analysis of FoxO1 protein levels. α-Tubulin was used as the loading control. (b) Quantification of relative FoxO1 protein levels with densitometry. ImageJ 1.42q software was used to analyze the density of each band, and the relative expression level was normalized to that of α-tubulin. Yellow bar, total FoxO1 protein. Green bar, endogenous FoxO1 protein. Orange bar, exogenous FoxO1 protein. (c) The apoptotic signals in MGCs after transfection were detected with FITC fluorescence (green), and the nuclei were counterstained with DAPI (blue). Bar, 20 μm. (d) Quantification of the apoptotic signals. The optical density was evaluated in each MGC with ImageJ 1.42q software. Experiments were performed in triplicate, and cells were counted in three randomly selected fields in each coverslip. Data represent the means±S.E. (e) Transcription changes in Bim in response to FoxO1 overexpression-induced apoptosis in MGCs. Expression data were normalized to that of β-actin. *P<0.05; **P<0.01, Student's t-testCC BY · PubMed Central
Figure 8 FoxO1 directly bound to the FRE site in the FoxO1 promoter. (a) A 2048-bp fragment of the FoxO1 promoter was amplified with PCR using mouse genomic DNA (bases 78–2125, 5'-UTR of FoxO1) containing a FRE (AGTAAACAAA) and cloned into the pGL3-Basic plasmid as pGL3-FoxO1 (W). pGL3-FoxO1 (M) was obtained by introducing four mutations (italics) into the FRE site. (b) FoxO1 reporter activities in mouse 3T3 cells co-transfected with FoxO1 expression vectors and FoxO1 promoter constructs for 36 h. The reporter activities were normalized to those of pRL-TK. RLA, relative luciferase activities. (c–e) The binding of FoxO1 to the FoxO1 promoter in MGCs or 3T3 fibroblasts was detected with ChIP assays following FSH treatment for 6 h. DNA was isolated from the precipitated complexes as a template for qRT-PCR (c). The qRT-PCR products were then analyzed on a 2% agarose gel (d) and quantified with densitometry using ImageJ 1.42q software (e). The amount of immunoprecipitated DNA for each ChIP reaction is shown as the percentage of input chromatin. IgG was the negative control. The data represent the means±S.E. (n=3). *P<0.05; **P<0.01, Student's t-test
Figure 8 FoxO1 directly bound to the FRE site in the FoxO1 promoter. (a) A 2048-bp fragment of the FoxO1 promoter was amplified with PCR using mouse genomic DNA (bases 78–2125, 5'-UTR of FoxO1) containing a FRE (AGTAAACAAA) and cloned into the pGL3-Basic plasmid as pGL3-FoxO1 (W). pGL3-FoxO1 (M) was obtained by introducing four mutations (italics) into the FRE site. (b) FoxO1 reporter activities in mouse 3T3 cells co-transfected with FoxO1 expression vectors and FoxO1 promoter constructs for 36 h. The reporter activities were normalized to those of pRL-TK. RLA, relative luciferase activities. (c–e) The binding of FoxO1 to the FoxO1 promoter in MGCs or 3T3 fibroblasts was detected with ChIP assays following FSH treatment for 6 h. DNA was isolated from the precipitated complexes as a template for qRT-PCR (c). The qRT-PCR products were then analyzed on a 2% agarose gel (d) and quantified with densitometry using ImageJ 1.42q software (e). The amount of immunoprecipitated DNA for each ChIP reaction is shown as the percentage of input chromatin. IgG was the negative control. The data represent the means±S.E. (n=3). *P<0.05; **P<0.01, Student's t-testCC BY · PubMed Central

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Metadata

Authors
Shen, M., Liu, Z., Li, B., Teng, Y., Zhang, J., Tang, Y., Sun, S.-C., & Liu, H.
Affiliations
  • Nanjing Agricultural University (CN)
  • Chinese Academy of Agricultural Sciences (CN)
Corresponding author
  • Sun, S-C
  • Liu, H
ORCID
Venue
Cell Death & Disease
Publisher
Springer Science and Business Media LLC
Published
2014-10-16
Volume
5
Issue
10
Pages
e1475-e1475
ISSN
2041-4889 (ISSN-L)
DOI
10.1038/cddis.2014.400
PMC
PMC4237239
OpenAlex
W2071278611
Cited by
123
References
66
Citations by year
2026: 7 · 2025: 16 · 2024: 17 · 2023: 11 · 2022: 12 · 2021: 13 · 2020: 14 · 2019: 4 · 2018: 9 · 2017: 10 · 2016: 6 · 2015: 4
FWCI
6.60
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Disease (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
National Key Research and Development Program of China (2014CB138502)
Topics
  • Health Sciences › Medicine › Reproductive Medicine › Reproductive Biology and Fertility
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › FOXO transcription factor regulation
  • Health Sciences › Medicine › Reproductive Medicine › Ovarian function and disorders
Keywords
FOXO1, follicle-stimulating hormone, granulosa cells, apoptosis, follicular atresia, positive feedback loop
MeSH terms
Forkhead Box Protein O1, Animals, Base Sequence, Cell Survival, Cells, Cultured, Female, Follicle Stimulating Hormone, Granulosa Cells, Mice, Inbred ICR, Molecular Sequence Data, Protein Binding, Signal Transduction, Down-Regulation, Apoptosis, Cyclic AMP-Dependent Protein Kinases, Cytoprotection, Phosphatidylinositol 3-Kinases, Proto-Oncogene Proteins c-akt, Forkhead Transcription Factors

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