Age-associated up-regulation of EGR1 promotes granulosa cell apoptosis during follicle atresia in mice through the NF-κB pathway

Abstract

Follicular atresia is the main process responsible for the loss of follicles and oocytes from the ovary, and it is the root cause of ovarian aging. Apoptosis of granulosa cells (GCs) is the cellular mechanism responsible for follicular atresia in mammals. Recent advances have highlighted fundamental roles for EGR1 in age-related diseases via the induction of apoptosis. In the present study, we found that the expression of EGR1 was significantly increased in aged mouse ovaries compared with young ovaries. Immunohistochemical analysis revealed strongly positive EGR1 staining in atretic follicles, especially in apoptotic granulosa cells. We further showed that EGR1 up-regulation in mouse primary granulosa cells inhibited cell proliferation and promoted apoptosis. In addition, the promotion of apoptosis in GCs by EGR1 increases over time and with reactive oxygen species (ROS) stimulation. Our mechanistic study suggested that EGR1 regulates GC apoptosis in a mitochondria-dependent manner and that this mainly occurs through the NF-κB signaling pathway. In conclusion, our results suggested that age-related up-regulation of EGR1 promotes GC apoptosis in follicle atresia during ovarian aging.

Keywords: EGR1; NF-κB; apoptosis; follicular atresia; granulosa cell; ovarian aging.

Figure 1.

Expression of EGR1 Increased as the Ovary Aged (A-C). (A) Expression levels of Egr1 mRNA in 6- and 36-week-old mouse ovaries were analyzed via quantitative RT-PCR. (B) Expression levels of EGR1 protein in 6- and 36-week-old mouse ovaries were analyzed with Western blotting. (C) Relative quantitation of the EGR1 protein expression shown in panel B. The relative protein levels were calculated from the band intensities. Triplicate tests were performed for each sample. *p < 0.05, **p < 0.01. The data in the lower panels represent the means ± SEM of 3 independent experiments. Expression of EGR1 in Mouse GCs Increased as the Follicles Grew and Became Atretic. The cellular localization of EGR1 in ovaries and the expression levels of EGR1 in follicles were measured using immunohistochemistry (D-K). (D) Negative control with PBS. (E) EGR1 expression in follicles at different stages (primordial follicles, red arrows; growing follicles, yellow arrows; antral follicles, green arrows; atretic follicles, purple arrows). (F) No distinct staining was observed in primordial follicles. (G) and (H) Slight staining in growing follicles. (H) and (I) Significant staining in antral follicles. (J) and (K) Strong positive staining in atretic follicles. Oocyte, red arrowheads; granulosa cells, yellow arrowheads; theca and interstitial cells, green arrowheads. Original magnifications: ×10 (D), ×20 (E, G, H and I), and ×40 (F, I and K).

Figure 2.

Up-regulation of EGR1 Significantly Inhibited GC Proliferation. (A) The expression levels of Egr1 mRNA in GCs at 48 h after transfection with Cmv6-Egr1 and vector were analyzed via quantitative RT-PCR. (B) The expression levels of EGR1 protein in GCs at 48h after transfection with Cmv6-Egr1 or vector were analyzed with Western blotting. (C) Relative protein levels as shown in panel B. (D) Proliferation of GCs in the Cmv6-Egr1, vector and control groups was measured using the EdU incorporation assay. The control group was only transfected with Lipofectamine 3000 and P 3000. Red fluorescence representes EdU-labeled GCs. (original magnification ×20). (E) The proportion of EdU-positive GCs as shown in panel D. (F) The protein expression level of P21, CYCLIN B1, and CYCLIN D1 in GCs after transfection with Cmv6-Egr1 or vector at 48 h were detected by Western blotting. (G) Relative protein levels as shown in panel F. *p < 0.05, **p < 0.01. The data are showed as the means ± SEM from 3 independent tests.

Figure 3.

Up-regulation of EGR1 Significantly Promoted GC Apoptosis. (A) The apoptosis rate of GCs transfected with Cmv6-Egr1 or vector at 48 h and 72 h was determined using FACS analysis. The horizontal axis represents Annexin V-FITC, and the vertical axis represents PI. Q1, Annexin V-FITC-/PI+: necrotic cells. Q2, Annexin V-FITC+/PI+: late apoptotic cells. Q3, AnnexinV-FITC+/PI-: early apoptotic cells. Q4, AnnexinV-FITC-/PI-: live cells. We used Q2+Q3 as the rate of apoptosis. (B) The apoptosis rate of GCs after transfection with Cmv6-Egr1 or vector, incubation for 48 h and then incubation with 200 μM H₂O₂ for 2 h was examined using FACS analysis. (C) The apoptosis rate as shown in panel A. (D) The apoptosis rate as shown in panel B. (E) Morphological changes in GCs were stained with Hoechst 33342 and evaluated using fluorescence microscopy. GCs transfected with Cmv6-Egr1 or vector and incubated for 48 h were then incubated with various concentrations of H₂O₂ for 24 h. (F) The apoptosis rate of GCs as shown in panel E. Original magnification ×40. *p < 0.05, **p < 0.01. The data are shown as the means ± SEM from 3 independent tests.

Figure 4.

Up-regulation of EGR1 Promoted GC Apoptosis through the Mitochondrial Pathway and Induced P53 Activation. (A) The expression levels of BCL2, CASPASE-9, CASPASE-3, Cleaved CASPASE-3 and Cleaved CASPASE-8 proteins in GCs transfected with Cmv6-Egr1 or vector at 48 h were determined with Western blot analysis. (B) Relative protein levels as shown in panel A. (C) The expression levels of PTEN, p-PTEN, P53, and p-P53 in GCs transfected with Cmv6-Egr1 or vector at 48 h were examined using Western blot analysis. (D) Relative protein levels as shown in panel C. *p < 0.05. The data in the right panels represent the means ± SEM of 3 independent experiments.

Figure 5.

Up-regulation of EGR1 Promotes Apoptosis in Granulosa Cells through NF-κB Signaling. (A) The expression levels of NF-κB and p-NF-κB in GCs transfected with Cmv6-Egr1 or vector at 48 h were examined using Western blot analysis. (B) Relative protein levels as shown in panel A. (C) The protein expression levels of NF-κB and p-NF-κB in 6-week and 36-week ovaries were tested via Western blotting. (D) Relative protein levels as shown in panel C. The data in the right-hand panels represent the means ± SEM of 3 independent experiments. *p < 0.05.