| Objective:Polycystic ovary syndrome(PCOS),the major cause of clinical anovulatory infertility,is a complex endocrine and metabolic disorder that affects approximately 6%～20%of women of reproductive age.PCOS have a heterogeneous at clinical presentation,and its main clinical characteristics include irregular menstruation,hyperandrogenic manifestations or hyperandrogenemia,and polycystic ovarian morphology on ultrasound.In addition to suffering from infertility,recent studies suggested that PCOS patients had a higher risk of long-term complications,including cardiovascular disease,hyperlipidemia,hypertension.However,up to now,the pathogenesis of PCOS is not well understood and many studies have suggested that the development of PCOS may be the result of a combination of genetic and environmental factors.Ovarian granulosa cells and follicular fluid,as important components of the ovarian microenvironment,play an essential role in follicular development and ovulation,providing the material basis and spatial support for oocyte growth and development.As a type of cells with secretory function in the ovarian microenvironment,ovarian granulosa cells can participate in the development of PCOS by proliferation,apoptosis and hormone synthesis.In recent years,with the advancement of proteomics technology,more and more researchers have focused on the role of proteomics in the pathogenesis of complex diseases.In modified proteomics,phosphorylation modifications of proteins are also known as "the switch of protein function".By combining phosphorylation proteomics and proteomics of target cells from control and disease groups,researchers are able to identify disease-specific proteins and phosphorylation sites.However,there are no studies on the proteomics of phosphorylation modifications in PCOS.Therefore,in Chapter Ⅰ,we focused on depicting the expression profile of phosphorylated proteomics and proteomics in ovarian granulosa cells of PCOS patients by performing 4D-label-free quantitative proteomics analysis.Based on the result of Chapter Ⅰ,in ChapterⅡ,we continue to explore the role and mechanism of the differentially expressed protein S100 calcium binding protein all(S100A11)in ovarian granulosa cells.In addition to granulosa cells,follicular fluid is also an important component of the ovarian microenvironment and plays an important role in maintaining normal ovarian physiology.Bile acid metabolites have been found in rat ovaries and components of the bile acid synthesis pathway have been detected in ovarian follicles.However,there are no studies related to bile acid metabolites in the follicular fluid of PCOS.In the Chapter Ⅲ,the bile acid metabolites in follicular fluid were identified in order to map the bile acid metabolism in PCOS follicular and provide new insight to reveal the pathogenesis of PCOS.Methods:In Chapter Ⅰ,a total of 39 control patients and 21 patients with PCOS were included.Follicular fluid was collected on the day of oocyte retrieval and ovarian granulosa cells were extracted from follicular fluid.Ovarian granulosa cells from the same group of patients were mixed and divided equally into triplicates.The proteomics and phosphorylation-modified proteomics were determined by 4D-label-free quantitative proteomics technology to depict differentially expressed proteins(DEPs)and differentially phosphorylated proteins(DPPs).DEPs and DPPs were analyzed by bioinformatics techniques.In Chapter Ⅱ,we validated the differentially expressed protein S100A11 identified in Chapter I with expanded sample.Correlation analysis was performed between the relative expression level of S100A11 and clinical baseline data of participants.Subsequently,by constructing PCOS animal models and overexpressing adenovirus,the role of S100A11 in ovarian granulosa cells and its mechanism were investigated.For in vitro functions,qRT-PCR,Western blot,Edu,flow cytometry,immunofluorescence,electron microscopy,Oil Red O staining and ROS kit were used to reveal the role and specific mechanisms of S100A11 in cell proliferation,apoptosis,cell cycle,subcellular localization,mitochondrial morphology,lipid droplet accumulation and ROS accumulation.In Chapter Ⅲ,a total of 66 participants were included,which comprised 35 PCOS patients and 31 control patients.Follicular fluid was collected,centrifuged and its supernatant stored for follow-up experiments.Bile acid metabolites in follicular fluid were detected and classified for analysis using ultra performance liquid chromatography/tandem mass spectrometry(UPLC-MS/MS).Pearson correlation analysis was performed between the quantitative results of differential bile acids and clinical indices to clarify the alterations of bile acid metabolites in follicular fluid in PCOS.Results:Among the proteomic results,293 up-regulated and 420 down-regulated DEPs were identified in the PCOS group.522 proteins had increased phosphorylation and 159 proteins had decreased phosphorylation in the PCOS group.Analysis of phosphorylation-modified sites identified 933 up-regulated phosphorylation sites and 211 down-regulated phosphorylation sites in the PCOS group.Bioinformatic analysis of DEPs and DPPs revealed that differences were enriched in the pathways of steroid biosynthesis,pentose phosphate pathway and vascular endothelial growth factor(VEGF)signaling pathway et al.Proteomic results suggested that protein S100A11 expression was elevated in PCOS ovarian granulosa cells.After expanding the clinical sample and constructing mouse model,we confirmed that S100A11 expression was elevated in both PCOS patients and PCOS animal models,and there was a positive correlation between the relative expression level of S100A11 and Anti-Mullerian hormone(AMH),testosterone(T),triglyceride(TG)and antral follicle count(AFC).Cellular experiments confirmed that S100A11 was mainly expressed in the nucleus of ovarian granulosa cells.Overexpression of S100A11 in ovarian granulosa cells increased the phosphorylation level of Dynamin-related protein 1(DRP1)serine(Ser)616 site,promoted mitochondrial fission,increased the level of reactive oxygen species(ROS)and lipid droplet accumulation in granulosa cells.In S100A11 overexpressing granulosa cells,Cytochrome C was released from mitochondria and Cleaved-Caspase 3 levels were increased,which contributed to the apoptosis of granulosa cell and the arrest of cell cycle in the G1 phase,further participating in the development of ovulation disorders in PCOS.Follicular fluid,another important component of the ovarian microenvironment,was detected for 24 bile acid metabolites.The level of total bile acid in the follicular fluid was not significantly different between two groups,but four differential bile acid metabolites were found to be significantly elevated in the PCOS group,namely glycocholic acid(GCA),taurocholic acid(TCA),glycocholic goose deoxycholic acid(GCDCA)and glucuronosyl ursodeoxycholic acid(CDCA-3Gln).GCDCA was positively correlated with the level of serum FSH and LH,the level of CDCA-3Gln was positively correlated with AFC,and there was no statistical correlation between other differential bile acid metabolites and clinical characteristics.Conclusions:In the study,the differences of proteomic and phosphorylated proteomic differences in ovarian granulosa cells from PCOS patients were revealed,and protein S100A11 expression increased in PCOS group.Elevated levels of DRP1 Ser616 phosphorylation and mitochondrial fission in S100A11 overexpressing granulosa cells,which in turn affect the function of ovarian granulosa cells,promote the development of PCOS.In addition,we have explored the bile acids metabolomics in follicular fluid of PCOS patients.However,individual differences in clinical samples cannot be ignored,and the role of bile acid metabolites in ovarian function in PCOS remains to be further investigated.In conclusion,based on proteomic and metabolomic analyses,this study investigated and explored the role of granulosa cells and follicular fluid in the PCOS ovarian microenvironment,providing a new perspective to reveal the pathogenesis of PCOS. |
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