The Role And Mechanism Of Lysine Succinylation,a Newly Post-translational Modification,in Ovarian Aging

Background and Objective : The research on the mechanism of ovarian aging is of great significance for female health and human reproduction.However,the molecular pathways involved in ovarian aging remain unclear.Protein post-translational modification(PTM)regulates cell function by affecting gene expression or protease activity,which plays an important role in the regulation of ovarian function and the occurrence and development of related diseases.Lysine succinylation(Ksuc),a newly PTM,has been found to be widely conserved in eukaryotic and prokaryotic cells and is associated with various physiological and pathological processes such as cell metabolism and tumorigenesis.Compared with traditional PTMs(such as acetylation and methylation),the larger molecular weight of succinyl group with two negative charges makes the greater influence of Ksuc on protein properties.Therefore,it is speculated that Ksuc may also exist in the ovary and play an important regulatory role,which is more promising for research.However,the role and mechanism of Ksuc in ovarian aging have not been reported so far.Therefore,this study focused on Ksuc to observe the distribution of Ksuc in mouse ovary;the levels of Ksuc in aged and young ovaries were detected;screening and validation of components of the Ksuc regulatory system related to ovarian aging,to explore the role and possible mechanism of Ksuc in the regulation of ovarian function and to provide new content for solving the problems related to ovarian aging.Methods:1.The distribution of Ksuc in mouse ovary was detected by tissue immunofluorescence(IF)staining.2.The pathological ovarian aging model was established by intraperitoneal injection with cytoxan and busulfan.3.Western blot(WB)and immunohistoc hemistry(IHC)staining of Ksuc were assessed in mouse aged and young ovaries.4.Quantitative real-time PCR(q RT-PCR)was used to screen and identify Ksuc candidate regulatory enzyme-KAT2 A in the mouse physiological ovarian aging model.5.QRT-PCR,WB and IHC were used to detect the levels of K AT2 A in mouse ovaries of different months.6.The co-localization of KAT2 A and Ksuc in mouse primary granulosa cells(GCs)was detected by cell IF staining.7.WB was used to detect the changes of Ksuc levels in the mouse ovarian tissue after sodium succinate(succinyl donor)administration,and the GCs knockout and overexpression of KAT2 A to verify that cofactors and KAT2 A were components of the Ksuc regulatory system in mouse ovary.8.The global Ksuc level in the mouse ovary was increased by in situ injection of sodium succinate into ovarian tissue.Then evaluating their effects on ovarian index,anti-Mullerian hormone(AMH)and estradial(E2),follicle number,ovarian cell proliferation and apoptosis,and molecular levels of proliferation-and apoptosis-related genes and aging marker P21.9.Small interfering RNA and overexpression lentiviral vector of K AT2 A were co-incubated with mouse primary GCs to evaluate the effects of Ksuc regulated by KAT2 A on proliferation,apoptosis and mitochondria-related functions of the cells.10.KAT2 A overexpression lentivirus vector was injected into mouse ovaries in situ to evaluate the effects of Ksuc regulated by KAT2 A on ovarian index,levels of anti-Mullerian hormone and estroge n,number of follicles,and effects of molecular levels related to apoptosis,oxidative stress and the aging marker P21 in ovarian cel s.Results:1.Ksuc is present in the mouse ovary and mainly occurs in the protein of 25-130 k Da.2.Excessive Ksuc was found in mouse physiologically(p < 0.01,v.s.3 M)and pathologically(p < 0.05)aged ovaries.3.Increased Ksuc with sodium succinate can reduce ovarian index(p < 0.05),AMH(p < 0.05)and E2(p < 0.01)levels,promote follicular atresia and GCs apoptosis of ovary in vivo.4.KAT2 A in mouse ovary showed an aging increasing trend.5.The imbalance of Ksuc level regulated by KAT2 A can promote apoptosis and mitochondrial function damage,and inhibit the proliferation of GCs cultured in vitro.6.Ksuc regulated by K AT2 A can reduce the ovarian index and AMH level,promote follicular atresia,and change the molecular levels of genes related to ovarian cell apoptosis,aging and oxidative stress of mouse ovary in vivo.7.Ksuc in mouse ovary can be regulated by succinyl donors(cofactors)and KAT2 A.Conclusions:1.Ksuc can be regulated by cofactors and KAT2 A in the mouse ovary.2.The mechanism of ovarian aging with elevated Ksuc may be associated with increased KAT2 A level,decreased proliferative capacity,increased apoptotic function and impaired mitochondrial function of GCs.