| Ⅰ.ObjectiveReproductive process is a complicated event which involves a variety of organs,including ovary,fallopian tubes,endometrium and embryo.The process of pregnancy needs to go through four stages: egg maturation,ovulation,fertilization and embryo implantation.The dysfunction of any of these could lead to infertility.Currently,infertility is common in women with reproductive age and the incidence is up to10%-12%.The reasons for infertility involved many aspects.For example,ovulation dysfunction(Polycystic ovary syndrome),tubal blockage,poor endometrial receptivity and failure of embryo implantation,etc.These dysfunctions may result in infertility or pregnancy related diseases.Polycystic ovary syndrome(PCOS)is a common reproductive disorder in women of childbearing age,characterized by anovulation,hyperandrogenism and polycystic ovary,which is the most common reason that causes female infertility.Ovary is an important female reproductive organ that producing kinds of hormones(progesterone,androgen,estrogen,luteinizing hormone and follicle stimulating hormone,etc)and cytokines(LIF),which plays an important role in ovulation,menstrual cycle and embryo implantation.Due to the heterogeneity of endocrine abnormalities and complexity of clinical manifestations in PCOS patients,the clinical diagnosis is usually depend on B-ultrasonography,and lack the specific serological markers for PCOS detection.Moreover,the treatment for PCOS in clinic is mainly steroid drugs andovarian surgery,and the steriod drugs for PCOS are accompanied with many side effects.Thus,it is necessary and significance to analyze the alterations of molecules in PCOS,and explore the pathogenesis comprehensively and systematically to find sensitive and specific serological biomarkers and non-steroidal hormone drugs to improve clinical detection,as well as to provide new treatment options for PCOS patients.PCOS and its reproductive dysfunctions are associated with endometrial receptivity and embryo implantation,which is less reported.The successful embryo implantation requires two conditions: good endometrial receptivity and mature embryo.The endometrium in the receiving state has the ability to receive mature embryo to implant.Mature embryos can adhere and invade the uterine endometrium in the recipient state to complete embryo implantation.The establishment of endometrium receptivity and embryo implantation are regulated by multiple hormones,expression and modifications of molecules.Glycosylation is an important post-translational modification of proteins that catalyzed by the specific glycosyltransferases.The fucosylated glycans which carried by glycoproteins are catalyzed by the specific fucosyltransferases,that regulated by hormones or cytokines.The alterations of the fucosyltransferases expression can lead to the changes of glycoproteins fucosylation modifications which affects cell functions.Studies have found that protein glycosylation could regulate ovarian functions,and mediated the identification and adhesion of maternal-fetal interface,as well as embryo implantation.It plays an essential role in reproductive process.It will lead to infertility or pregnancy failure when the endometrial receptivity goes down or failure to implantation caused by the alterations of protein glycosylation.In this study,we used clinical samples,cells and animal models to investigate the expression and regulation of reproductive hormones-α1,3 fucosylation-uPAR of ovarian-endometrium-embryo axis during the occurrence and development of PCOS,as well as the relationship with reproductive functions,and provide novel evidence and theoretical basis for clinical diagnosis and treatment of PCOS.II.Methods(I)The role and regulatory mechanism of reproductive hormones in PCOS1.Regulation of progesterone on the expression of proteins in serum and granulosa cells of PCOS patients(1)Differentially expressed serum proteins screened by protein array.The expression of five low abundance serum proteins(EREG,inhibin βA,IDE,PDGF-D and KNG1)was detected by ELISA and Western blot,and the correlation with progesterone was analyzed;(2)The cell proliferation ability was detected by CCK8 and colony formation assay,cell apoptosis ability was detected by TUNEL,DAPI staining and Western blot.2.The inhibitory effect of DHEA on EMT of ovarian granulosa cells through downregulating uPAR(1)Serum level of uPAR and testosterone in healthy women and PCOS patients was detected by ELISA,and the correlation between uPAR and testosterone was analyzed;(2)The cell proliferation ability was detected by CCK8,the migration and invasion abilities were detected by Transwell.The expression and activities of MMP2/9 were detected by Western blot and gelatin zymography;(3)The changes of cell morphology were observed under a microscope,the expression of EMT markers,uPAR,suPAR and the activation of PI3K/Akt,as well as MAPK signaling pathways were detected by Real-time PCR,Western blot and immunofluorescence;(4)DHEA induced PCOS rat model was used,the expression of uPAR,PCNA and EMT markers in ovary tissues of rats were detected by immunohistochemistry.3.Effect of ANP on the proliferation and apoptosis of ovarian granulosa cells through promoting the formation of NPRA/PGRMC1/EGFR complex(1)Level of ANP and hormones in serum was detected by ELISA,and the expression of ANP in ovary tissues was detected by Real-time PCR andimmunohistochemistry.The microvilli on the surface of endometrium was observed by scanning electron microscope.The expression of NPRA/C and PGRMC1 was detected by immunofluorescence,Real-time PCR,Western blot and immunohistochemistry;(2)Level of testosterone,progesterone and estradiol in culture medium was detected by ELISA.The cell proliferation ability was detected by CCK8,colony formation assay,Western blot and immunofluorescence,the cell apoptosis ability was detected by TUNEL,DAPI staining and Western blot;(3)The expression of PGRMC1,NPRA,as well as PCNA and the activation of MAPK/ERK signaling pathway was detected by co-immunoprecipitation and Western blot;The expression of PCNA and AP1(p-c-Fos and p-c-Jun)was detected by immunohistochemistry and Western blot.(Ⅱ)Regulation of α-1,3 fucosylation on the formation of endometrial receptivity(1)Serum level of miR-200 family members(miR-200 a,miR-200 b,miR-200 c,miR-141 and miR-429)was detected by Real-time PCR.The diagnostic values were measured by ROC curve,and the correlation of FUT4 and mi R-200 c was analyzed;(2)The cell proliferation ability was detected by CCK8 and colony formation assay.The microvilli on cell surface was observed by scanning electron microscope.The adhesion ability of Ishikawa cells was detected by cell adhesion assay;(3)The binding sites of miR-200 c and FUT4 were predicted by software.The changes of luciferase activity were detected by dual luciferase gene report assay.The expression of FUT4 was detected by Real-time PCR,Western blot and immunofluorescence;(4)The expression of α-1,3 fucosylation(LTL,LeY),as well as Wnt/β-catenin signaling pathway was detected by Western blot and co-immunoprecipitation;(5)The number of embryo implantation was counted on day 9 of pregnancy by using mouse early pregnancy model.The expression of miR-200 c,FUT4,LTL and LeY in endometrium was detected by Real-time PCR,fluorescence in situ hybridization and immunofluorescence.The microvilli and pinosome on the surface of endometrium were observed by scanning electron microscope.The expression of PCNA and Wnt/β-cateninsignaling pathway was detected by immunohistochemistry.(III)Study of uPAR promoting embryo implantation1.Expression of uPAR on human trophoblast cells and its role on trophoblast invasion(1)The expression of uPAR was detected by immunohistochemistry,Western blot,Real-time PCR and immunofluorescence;(2)The migration and invasion abilities were detected by villus in vitro culture and Transwell.The activities of MMP2/9 were detected by gelatin zymography.The expression of TIMP1/2 was detected by Western blot.The cell proliferation ability was detected by CCK8,and the cell apoptosis ability and uPAR expression were detected by Western blot.2.uPA/uPAR promotes trophoblast invasion through upregulating FUT4(1)The expression of AP1 and MAPKs signaling pathways was detected by immunohistochemistry,Western blot and immunofluorescence;(2)The expression of uPAR,FUT4 and Le Y was detected by Real-time PCR,Western blot and immunofluorescence;(3)The binding of AP1 to FUT4 was detected by EMSA and ChIP assays;(4)The cell migration and invasion abilities were detected by Transwell.III.Results(I)The role and regulatory mechanism of reproductive hormones in PCOS1.Regulation of progesterone on the expression of proteins in serum and granulosa cells of PCOS patients(1)The expression of progesterone and serum proteins in patients of PCOS and healthy women were different.The level of progesterone,EREG,inhibin βA,IDE,PDGF-D and KNG1 in serum of PCOS patients was decreased significantly,and positively correlated with progesterone;(2)Progesterone upregulated the expression of EREG,inhibin βA,IDE,PDGF-D and KNG1 in ovarian granulosa cells.EREG and inhibin βA promoted the proliferation,and inhibited the apoptosis of ovarian granulosa cells.2.The inhibitory effect of DHEA on EMT of ovarian granulosa cells through downregulating uPAR(1)The expression of uPAR in serum of PCOS patients was decreased;(2)DHEA inhibited the proliferation,migration,invasion and EMT through downregulating the expression of uPAR/suPAR and inactivation of PI3K/Akt and MAPK signaling pathways.3.Effect of ANP on the proliferation and apoptosis of ovarian granulosa cells through promoting the formation of NPRA/PGRMC1/EGFR complex(1)The expression of ANP in PCOS patients and PCOS rats was decreased,and ANP treatment improved the ovarian morphology and functions;(2)RU486 inhibited the proliferation and promoted the apoptosis of ovarian granulosa cells through downregulating the expression of PGRMC1;(3)ANP promoted the proliferation and inhibited the apoptosis of ovarian granulosa cells through upregulating the expression of NPRA/C;(4)ANP promoted the proliferation of ovarian granulosa cells through regulating the formation of NPRA/PGRMC1/EGFR complex and activated MAPK/ERK signaling pathway and AP1.(Ⅱ)Regulation of α-1,3 fucosylation on the formation of endometrial receptivity(1)The expression of miR-200 family members in serum of infertility and abortion patients was increased,while FUT4 was decreased,and they were negatively correlated in serum of infertility patients;(2)miR-200 c inhibited the proliferation and adhesion of endometrial epithelial cells;(3)FUT4 was a novel target of miR-200c;(4)miR-200 c inhibited α-1,3 fucosylation on CD44,and inhibited the proliferation and adhesion of endometrial epithelial cells through suppressing the activation of Wnt/β-catenin signaling pathway;(5)miR-200 c inhibited the formation of endometrial receptivity and embryo implantation by regulating FUT4/α-1,3 fucosylation in vivo.(Ⅲ)Study of uPAR promoting embryo implantation1.Expression of uPAR on human trophoblast cells and its role on trophoblast invasion(1)The expression of uPAR in placental villi of pregnancy women was higher than threatened abortion patients;(2)uPAR si RNA inhibited the proliferation,migration and invasion of trophoblast cells;(3)LIF promoted trophoblast invasion through upregulating the expression of uPAR and activated the PI3K/Akt signaling pathway.2.uPA/uPAR promotes trophoblast invasion through upregulating FUT4(1)The expression of AP1 in placental villi and serum of normal pregnancy women was higher than that in threatened abortion patients;(2)uPA/uPAR promoted the expression of p-c-Fos and p-c-Jun through activating JNK signaling pathway;(3)uPA/uPAR promoted the migration and invasion of trophpblast cells through activating AP1 and upregulating FUT4.Ⅳ.Conclusion1.The progesterone and serum proteins in PCOS patients and healthy women were differentially expressed,and progesterone upregulated the expression of EREG and inhibin βA which could apply as potential serum biomarkers for PCOS patients with low progesterone level;2.DHEA inhibited the proliferation,migration,invasion and EMT of ovarian granulosa cells through downregulating the expression of uPAR/suPAR and inactivated the PI3K/Akt and MAPK signaling pathways;3.ANP improved ovarian functions through regulating the formation of NPRA/PGRMC1/EGFR complex,as well as activating MAPK/ERK signaling pathway and AP1;4.The formation of endometrial receptivity was correlated with α-1,3 fucosylation;5.miR-200 c inhibited the formation of endometrial receptivity through targetingFUT4 and α-1,3 fucosylation on CD44;6.miR-200 c and FUT4 could be served as potential markers for endometrial receptivity,as well as the diagnostic and therapeutic targets for infertility;7.uPAR was expressed on embryo trophoblast cells;8.LIF promoted trophoblast migration and invasion through upregulating the expression of uPAR;9.uPA/uPAR promoted trophoblast invasion via activating AP1 and increased the expression of FUT4. |
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