The Role Of EGR1-regulated Granulosa Cell Apoptosis In Ovarian Aging And Its Mechanism

Background and purposes:Ovarian aging is a process in which female ovarian function declines with age and is affected by natural,genetic,genetic,environmental,immune and artificial factors,and it can affect multiple organs of the body.The decline of fertility caused by its reproductive function aging and the reduction of estrogen secretion and the resulting diseases such as perimenopausal symptoms,osteoporosis,urogenital atrophy symptoms and cardiovascular disease caused by its endocrine function aging,bring great harm to women’s physical and mental health.Follicle 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 is the cellular mechanism responsible for follicular atresia in mammals.The aim of this study is to explore the expression manner of EGR1 in ovary during ovarian aging and to investigate the effect of Egrl on the proliferation and apoptosis of granulosa cell and its mechanisms.Methods:(1)The expression of EGR1 in the ovaries of young and aged mice and the accelerated ovarian aging model and the calorie restriction(CR)ovarian aging delayed model mice were determined by real-time quantitative PCR and Western blot;The expression and location of EGR1 in ovary and follicles was determined by the immunohistochemical method.(2)The expression of EGR1 in primary granulosa cells of 3-week-old mice was upregulated in vitro using an Egrl-overexpressing plasmid.The up-regulation of EGR1 in granulosa cells after transfection was detected by real-time quantitative PCR and Western blot.The effect of EGR1 up-regulation on the proliferation of granulosa cells was detected by EdU assay.The effect of EGR1 up-regulation on granulosa cell apoptosis was detected by flow cytometry combined with Annexin V/PI staining and Hoechst staining.The effect of EGR1 up-regulation on the proliferation-related genes in granulosa cells was detected by real-time PCR and Western blot.The effect of EGR1 up-regulation on the expression and activity of apoptosis-related proteins was detected by Western blot.(3)The molecules of related signaling pathway regulated by EGR1 up-regulation in granulosa cells and the same molecules’ change in aged mice was detected by Western blot.Results:(1)The expression of EGR1 mRNA and protein was significantly higher in the ovary of aged mice,compared with that of the young mice;and the expression of EGR1 protein in the ovary of the ovarian aging model mice was also significantly higher than that of the control group.The EGR1 protein expression in the oocytes was observed simultaneously in follicles at different stages,but no significant difference was found.In addition,there was no EGR1 protein expression in the theca or in interstitial cells.Further,EGR1 staining was not observed in the cytoplasm of granulosa cells in primordial follicles.As the follicles grew,the EGR1 protein expression level was increased in granulosa cells.Almost all of the atretic follicles showed the strongest staining.(2)The proliferation of granulosa cells with EGR1 up-regulation was markedly lower than that of vector group,and so was the genes related to granulosa cells proliferation;The apoptotic rate of of granulosa cells with EGR1 up-regulation was significantly higher than that of vector group,and the apoptotic rate was dramatically elevated as the time extended and with ROS stimulation.And the expression of mitochondrial apoptosis-related proteins in granulosa cells changed in the EGR1 up-regulation group,while the expression of death receptor pathway-related proteins not.(3)The expression level of p-P53 in granulosa cells was significantly increased in the EGR1 up-regulation group.In the EGR1 up-regulation group,the expression level of p-NF-kB was significantly increased compared with the vector group.In accordance with our in vitro results,ovaries from 36-week-old mice displayed significantly increased p-NF-kB levels when compared with ovaries from 6-week-old mice.Conclusions:(1)EGR1 is associated with ovarian aging and plays an important role in follicular atresia and granulosa cell apoptosis.(2)Up-regulation of EGR1 inhibits proliferation and promotes apoptosis of granulosa cells.And the role of EGR1 in promoting granulosa cells apoptosis becomes more pronounced as time extends and with ROS stimulation.EGR1 promotes granulosa cells apoptosis through the mitochondrial dependent pathway.(3)EGR1 may inhibit proliferation of granulosa cells by activating phosphorylation of P53,and promotes apoptosis of granulosa cells through NF-κB signaling pathway,thereby promoting the occurrence of folliculer atresia.In summary,our study suggests that age-associated upregulation of EGR1 inhibits proliferation of granulosa cells by regulating P53 activity,and promotes apoptosis of granulosa cells through the activation of NF-κB pathway.Moreover,it can amplificate the injury of harmful stimuli such as ROS on the ovary,thereby promoting follicle atresia and regulating the process of ovarian aging.