Analysis Of The Underlying Etiology Of Early-onset Ovarian Insufficiency

Part Ⅰ Analysis of TBP2 gene in Chinese women with idiopathic premature ovarian insufficiencyObjective:It has previously been reported that TBP2 gene expressed exclusively in ovary and female TBP2 null mice were sterile due to defective folliculogenesis.We aimed to analyze the association between TBP2 gene and idiopathic POI in a Chinese Han population.Methods:A total of 60 idiopathic POI patients and 60 healthy controls were recruited in our study.Genomic DNA from peripheral leukocytes was extracted by QIAamp DNA Blood Mini Kit.All TBP2 exons were amplified by PCR and then analyzed by direct sequencing in all subjects.Chi-square was used to assess the genotype-distribution and allele frequency between cases and controls.Results:A known SNP(rs8019270)located in exon 1 was detected in both POI and control groups.There was no deviation from the Hardy-Weinberg equilibrium in rs8019270.Genotype distribution or allelic frequencies showed no difference between two groups(p>0.05).In addition,no plausible pathogenic mutation was identified.Conclusions:Our study suggested that TBP2 gene was not associated with the development of idiopathic POI in Chinese Han population.This result might be caused by our limited sample size and single ethnicity.Considering its function on mice fertility,further study in a larger sample is needed.Part Ⅱ The role and mechanism of miR-125a-5p in the pathogenesis of cisplatin-induced premature ovarian insufficiencySection Ⅰ Evaluation of mouse model of cisplatin-induced premature ovarian insufficiency and the miR-125a-5p level Objective:We aimed to establish mouse model of premature ovarian insufficiency by cisplain and evaluate miR-125a-5p and miR-125b-5p levels in mouse model.Methods:ICR mice were intraperitoneally injected with 3.0mg/kg cisplatin to establish mouse model of POI and injected with NaCl as control group.The body weight,estrous cyclicity,ovary weight and ovarian coeffcient were measured.ELISA was used to analyze E2,FSH and AMH level.Pathological changes of ovary,heart,kidney,liver and lung were observed using HE staining.The level of miR-125a-5p and miR-125b-5p were tested by Realtime PCR.Results:Compared with control group,the body weight was declined at day 4 after cisplatin injection and the estrous cycle was irregular.Ovary weight and ovarian coefficient were decreased(p<0.01).Cisplatin significantly reduced E2 and AMH level(p<0.01)and enhanced FSH level(p<0.01).The ovaries of cisplatin-induced POI mice were smaller and showed irregular structure.Heart,kidney,liver and lung appeared normal in both groups.The miR-125a-5p level in peripheral blood and ovaries from cisplatin-induced POI mice were significantly elevated(p<0.01),while miR-125b-5p showed no difference between two groups(p>0.05).Conclusions:Our study demonstrated that cisplain intraperitoneal injection was successful to establish mouse model of chemotherapy-induced POI,which could be a useful model for studying iatrogenic POI.In addition,miR-125a-5p might be associated with the development of chemotherapy-induced POI.Section Ⅱ The role and mechanism of miR-125a-5p in cisplatin-induced apoptosis of mouse granulosa cellObjective:Chemotherapy is a major reason for POI.Granulosa cells(GCs)apoptosis play an important role in follicle depletion.MicroRNAs are expressed abundantly in GCs and may be responsible for associated follicular atresia.We aimed to identify the role and mechanism of miR-125a-5p in cisplatin-induced apoptosis of mouse GCs(mGC).Methods:Cultured mGCs were treated with cisplatin to establish an in vitro POI cell model.Cell viability was evaluated by Cell Counting Kit-8 assay(CCK-8).The apoptosis was assessed by flow cytometry and cleaved caspase-3 protein expression.Estradiol and progesterone concentrations were measured by radioimmunoassay.Realtime PCR was used to analyze the level of miR-125a-5p and Western blot to measure STAT3 and p-STAT3 expression.The cell apoptosis was evaluated after transfected with miR-125a-5p MI and siRNA-STAT3 by flow cytometry and Western blot.The rescue experiment was conducted with miR-125 a-5p MI and GV230-STAT3/GV230-control.The relationship between miR-125a-5p and STAT3 was evaluated by luciferase reporter assays and Western blot in HEK293T or mGCs,respectively.Results:Cisplatin reduced mGC viability in a time-and dose-dependent manner.After treated with 10μg/ml for 24h,the percentage of apoptotic cells and cleaved caspase-3 were increased(p<0.01).In addition,estradiol,progesterone and STAT3/p-STAT3 levels were decreased(p<0.01).MiR-125a-5p was significantly higher in cisplatin-treated mGCs(p<0.05).Increased cell apoptosis and enhanced cleaved caspase-3 level were observed after transfecting with a miR-125a-5p MI or a siRNA-STAT3(p<0.01).Luciferase reporter assays showed that miR-125a-5p binds to the 3’untranslated region(UTR)of STAT3 gene(p<0.01).STAT3/phospho-STAT3 protein expression were downregulated by miR-125a-5p MI,and vice versa(p<0.01).However,miR-125a-5p did not affect the level of STAT3 mRNA(p>0.05).Conclusions:MiR-125a-5p promoted the apoptosis of mGC by targeting STAT3.Our finding implied that miR-125a-5p might play an important role in the pathogenesis of POI.Part Ⅲ The role of BPTF gene regulating Treg cells in the immune factors of premature ovarian insufficiencyObjective:Autoimmune oophoritis(AO)displayed ovaries infiltrating with lymphocytes and plasma cells,which is a cause of autoimmune POI.The role of cell immunity in the pathogenesis of POI has gained more attention nowadays.Thymocytes are diverted into Treg cells,which have potent function of immunosuppression.Treg cells belong to CD4+T cells.Aberrant number or function of Treg cells cause autoimmune diseases.A number of studies have showed the role of Treg cells in AO mouse model and POI patients.BPTF gene is involved in the development of early embryo and thymocytes.Our study aimed to identify the role of BPTF in regulating Treg cells,which might imply the immune factors in the pathogenesis of POI.Methods:The number of CD4+T,CD8+T and Treg cells,and the ratio of Treg/CD4+T,CD44highCD62low/CD4+T and CD44highCD62low/CD8+T in the thymus,spleen and lymph nodes were analyzed by flow cytometry.In addition,PD-1,CTLA-4 and CD25 expression on Treg cells isolated from both groups were analyzed,WT Treg cells and BPTF-KO Treg cells were sorted from FGC:Bptffl/wt and FGC:Bptfl/fl mice.Tresp cells were sorted from wildtype C57BL/6 mice by flow cytometry.Suppression assay was conducted by mixing different amounts of Treg cells and Tresp cells labeled with CFSE after 72h poststimulation.Lymphocytes were isolated from ovaries and assessed by flow cytometry.Results:,The CD4+T and CD8+T cells distribution appeared comparable between FGC:Bptfl/wt and FGC:Bptfl/fl mice(p>0.05).Compared with FGC:Bptffl/wt mice,Treg cell populations significantly reduced in the spleen and lymph nodes of FGC:Bptffl/fl mice(p<0.05),but showed no difference in the thymus(p>0.05).The ratio of CD44highCD62low/CD4+T cells in the spleen and lymph nodes and CD44highCD62low/CD8+T cells in the spleen of FGC:Bptffl/fl mice were higher(p<0.05),but there was no difference of CD44highCD62low/CD8+T cells ratio in the lymph nodes(p>0.05).Whereas BPTF-deficinet Treg cells in FGC:Bptffl/fl mice expressed normal levels of PD-1,CTLA-4 and increased level of CD25,BPTF-KO Treg cells showed reduced suppressive activities.In addition,the number of infiltrated CD4+and CD8+T cells in the ovary were significantly increased(p<0.01 and p<0.05,respectively).Conclusions:The study demonstrated that BPTF deficiency might cause aberrant T cell activation,dysfunction of Treg cells and infiltrating T cells in the ovary,which might contribute to AO.Our study implied that BPTF deficiency might be associated with autoimmune POI.However,in vivo experiment will be required to evaluate the ovarian function,which might be helpful to explicit the role of BPTF in POI.