The Mechanism Of Theaflavin-3＿3’-digallate In The Regulation Of Ovarian Aging

Ovarian aging refers to the gradual decline of ovarian function with the increase of women’s physiological age,which is manifested by a decrease in ovarian reserve,an increase in related aging markers,and a decrease in oocyte quality.With the postponement of women’s reproductive age and the intensification of the global population aging,the demand to delay ovarian aging to maintain fertility and improve women’s quality of life is imminent.Theaflavin 3,3’-digallate(TF3)is a naturally active tea polyphenolic compound extracted from black tea,and its antioxidant properties play an important role in maintaining human health and delaying aging,but the effect of TF3 on female reproduction in the process of aging remains unclear.Thus,this study is to evaluate the effect of TF3 on ovarian aging in the following 3 parts:1.Theaflavin-3-3’-digallate delays ovarian aging in aged mice.2.Theaflavin-3＿3’-digallate ameliorates oxidative stress damage in human ovarian granulosa cells by regulating SESN3.3.Theaflavin-3-3’-digallate protects postovulatory oocyte from aging.Part1 Theaflavin-3-3’-digallate delays ovarian aging in aged miceObjective:To investigate whether theaflavin-3＿3’-gallate supplementation delays ovarian aging in aged mice.Methods:9-month-old CD1 mice were fed with TF3(every two days)for 3 months until 12-month-old to evaluate the protective effect of TF3 on ovarian aging in mice.The mouse ovarian tissue was collected,and the mouse ovarian morphology,number of follicles,estrous cycle and serum hormone levels were detected to study the effect of TF3 treatment on ovarian reserve.In vitro fertilization(IVF)evaluated the effect of TF3 on embryonic development potential,immunofluorescence staining of MII oocyte and reactive oxygen species(ROS),glutathione(GSH),mitochondrial membrane potential(JC-1)staining to explore the effect of TF3 on egg quality.Masson staining was used to detect the degree of ovarian fibrosis,and oil red O staining was used to detect the distribution of ovarian lipid droplets.The Western-blot method detected protein expression in mice ovaries.Results:Compared with the aged group,TF3 supplementation in mice can significantly increase ovarian reserve,shorten the estrus cycle,and increase the number of litters in aged mice.The analysis of ovarian aging-related indicators found that TF3 can reduce the expression level of aging and ovarian aging marker genes in the adipose tissue around the ovaries.TF3 treatment significantly improved the number of MII oocyte.ROS and GSH staining of oocytes found that the TF3 group significantly reduced the ROS levels and increased GSH levels.At the same time,the proportion of abnormal spindle in the TF3 treatment group also decreased significantly.Conclusion:This study showed that TF3 can be used as a potential dietary supplement to improve ovarian function and oocyte quality in aged mice and maintain follicle pool.TF3 plays an important role in delaying ovarian aging and maintaining ovarian function in mice.Part 2 Theaflavin-3＿3’-digallate ameliorates oxidative stress damage in human ovarian granulosa cells by regulating SESN3Objective:To investigate whether theaflavin 3,3’-digallate is involved in the antioxidant stress damage of human ovarian granule cells and its mechanism.Methods:We collected and isolated human primary granulosa cells of female patients undergoing assisted reproductive technology in our center and used the human ovarian granule cell line KGN for in vitro culture.1mM H2O2 was used to construct cellular senescence and oxidative stress model.Flow cytometry was used to detect apoptosis rate and ROS levels,high-throughput second-generation RNA sequencing to detect the changes in gene expression with or without TF3.Small interfering RNA(small interfering RNA,siRNA)specifically knocked down endogenous SESN3 and AMPK genes,qRT-PCR detected the expression levels of related genes,and the Western-blot method detected the expression levels of related proteins.Results:TF3 treatment on granulosa cells downregulated oxidative stress damage and apoptosis levels in the aging model,high-throughput sequencing results showed that TF3 upregulated 459 genes,gene set enrichment analysis(GSEA)found that TF3 was significantly enriched in autophagy function.After specific knockdown of SESN3,it was found that the protective effect of TF3 on oxidative stress damage was disappeared and dependent on the AMPK-mTOR signaling pathway.Conclusion:TF3 protects against oxidative stress damage to human ovarian granule cells by upregulating SESN3 through AMPK-mTOR signaling pathway.Part 3 Theaflavin-3-3’-digallate protects postovulatory oocyte from agingObjective:To explore whether theaflavin 3,3’-digallate delays postovulatory oocyte from aging.Methods:We used 8-week-old CD1 mice to collect MII oocytes,and TF3 culture with a final concentration of 5 μM was added to M16 droplets for 24 hours to establish an oocyte aging model and supplemental TF3 model.Parthenogenesis was performed using 6-dimethylaminopurine(6-DMAP)to detect the evaluate the effect of TF3 on the developmental potential.Immunofluorescence staining and ROS,GSH,JC1 staining explored the effect of TF3 on oocyte quality.RT-qPCR detected mRNA changes in mitochondrial respiratory chains(MRC)and antioxidant genes.Results:Aging oocytes in vitro can cause increased levels of oxidative stress,decreased mitochondrial membrane potential,and abnormal mitochondrial respiratory chains that affect oocyte quality and embryonic outcomes.Supplementation with TF3 can effectively reduce the adverse effects caused by oxidative stress,increase the expression of antioxidant genes and the expression of genes related to the mitochondrial respiratory chain,thus delay the aging of oocytes in vitro and improve embryonic development outcomes.Conclusion:Our results showed that TF3 can inhibit oxidative stress-induced DNA damage,reduce ROS during the process of aging,improve oocyte quality and embryonic development potential,thereby provide a potential strategy for delaying postovulatory aging and improving the IVF outcomes.