Regulation Of Wnt Signaling Pathway In Oocyte Maturation Of PCOS Patients During Super Long Down-regulation Protocol

BackgroundPolycystic ovary syndrome(PCOS)is a common endocrine disorder in women of childbearing age.It is also the most common cause of infertility in women.The etiology and pathogenesis of PCOS have not yet been clear.The current research focuses on genetic related genes,environmental factors,and local molecular biological changes in the ovary.More and more evidence suggests that abnormal follicle development in PCOS patients leads to low oocyte potential,and follicular dysplasia is the main pathophysiological feature of PCOS.We have found in our clinical practice that PCOS patients with ovulation induction during follicular phase can obtain higher oocyte maturation rate,fertilization rate and high-quality embryo rate.It is speculated that after ovulation induction,ovarian maturation and subsequent IVF outcome may be improved by regulating certain signaling pathways.Cumulus granulosa cells(CCs),closely related to the oocyte,is closely associated with oocyte development,catabolizing cholesterol,glucose and other substances,providing energy by gap junctions around the oocytes.Linkage between CCs and oocytes plays an important role in regulating oocyte development and maturation.The protein encoded by the Wnt gene is a class of secretory glycoproteins,which not only plays a role in autocrine regulation with the membrane receptors of their own cells,but also in paracrine functions with membrane receptors of adjacent cells.It activates intracellular signaling pathways and regulates the expression of target genes,proliferation,differentiation,migration,polarity and apoptosis of the cells.The classical Wnt signaling pathway is highly conserved in different species.Molecular mechanism:receptor of Wnt/beta-catenin signal molecule is a curl protein(Frizzled),and its auxiliary receptor is low density lipoprotein receptor related protein 5/6(LRP5/6).Beta-catenin is the downstream signaling molecule of the Wnt/beta-catenin signaling pathway.When Wnt signaling pathway specific inhibitors exist outside the cell,the binding of Wnt protein and its receptor will be inhibited.The beta-catenin in the cytoplasm is combined with the degrading complex formed by the colon adenocarcinoma polypoin(APC),the glycogen synthetase-3 beta(GSK-3 beta)and the axial protein(Axin)three.At the same time,the degradation complex is found.The GSK-3 beta can phosphorylate the beta-catenin,and the beta-catenin after phosphorylation is degraded by the proteasome through the ubiquitination pathway,resulting in a significant decrease in the level of beta-catenin in the cytoplasm.When there is no Wnt signal pathway inhibitor,Wnt protein is combined with Frizzled and LRP5/6 receptors on the cell membrane,and then inhibits the degradation of beta-catenin in the cytoplasm by two effects,when beta-catenin reaches a certain amount,it is associated with the transcription factor TCF/LEF(T-cell factor/lymphocyte enhancer binding factor)family and other transfers.The transcriptional activation complex is formed by the combination of coactivator.As a transcription activator,the complex can induce the expression of the target gene of the Wnt/beta-catenin signaling pathway and thus play a regulatory role.Earlier studies found that abnormal oocyte development in PCOS patients was related to the Wnt/beta-catenin signaling pathway,and it was separated to the genes corresponding to the granulosa cells in normal people at the nuclear maturation stage.It was presumed that the Wnt/beta-catenin signaling pathway may play an important role in the regulation of the oocyte maturation in PCOS patients.In order to investigate the regulatory role and mechanism of Wnt/beta-catenin signaling pathway in oocyte maturation of PCOS patients during follicular phase of super long down-regulation protocol,we have designed and completed the following three experiments:Part ?:A new protocol of IVF in PCOS patients—super long down-regulation protocol during follicular phaseobjective:To evaluate the efficacy of specific in vitro fertilization(IVF)protocols on the patients with polycystic ovary syndrome(PCOS),and therefore analyze the first-rank intention IVF protocol.Method:408 patients with PCOS(464 treatment cycles)were divided into 3groups:IVF1 group(OC regimen,n=91),IVF2 group(GnRH-antagonist regimen,n=80),IVF 3 groups(Super-long drop regulation regimen,n=293),their IVF outcome were evaluated.Result:1.The number of eggs,oocyte maturation rate and high-quality embryo rate in the super long down-regulation group were significantly higher than those in the other two groups,the fertilization rate and cleavage rate of the three groups have no significant difference.2.After fresh embryo transplantation,the pregnancy rate of super long down-regulation group is significantly higher than that of other groups.Conclusion:This study showed that the super long down-regulation in follicular phase regimen had the advantages of simple treatment process,high oocyte maturation rate,high quality embryo rate and pregnancy rate.It is a good choice for PCOS patients to promote ovulation during IVF.Part ?:Regulation of Wnt signaling pathway in oocyte maturation of PCOS patientsChapter 1:Wnt/p-catenin signal transduction during follicualr development in miceMethod:1.Sexual maturity FVB and ICR female mice was executed by cervical dislocations.2.Take the ovaries and shave off the excess fat on the ovarian surface and put them in the 1.5ml EP tube containing PBS.3.Extraction of tissue protein.4.Immunohistochemical localization of LRP5/6,GSK3?,?,DVI3 and TCF-4 proteins in mouse ovarian tissue.5.Western Blot was used to detect the expression of LRP5/6,GSK3?,?,DVI3 and TCF-4 proteins.Result:1.Immunohistochemical localization of protein in mouse ovarian tissue(1)Immunohistochemistry was used to detect the localization of LRP5/6,GSK3?,? proteins in mouse ovarian tissues.LRP5/6 protein was highly expressed in the membrane of cells,and GSK3?,? protein was expressed in the cytoplasm of cells.(2)Immunohistochemistry was used to detect the localization of TCF-4 and DVI3 proteins in mouse ovarian tissues.DVI3 protein was highly expressed in the cell membrane and TCF-4 protein was highly expressed in the nucleus.2.Western Blot was used to detect the expression of proteins.(1)The expression of DVI3 protein in FVB mouse ovary was higher than that in ICR mouse ovary.(2)The LRP5/6 protein in mouse ovary was not detected by Wb assay,and the result was not obviously changed after phosphorylation of LRP5/6 protein.(3)Western Blot was used to detect TCF-4 protein in mouse ovary.TCF-4 protein was highly expressed in FVB mouse ovary,but it was low in ICR mouse ovary.Conclusion:1.IHC results showed that LRP5/6 protein was highly expressed in the membrane of cells,DVI3 was highly expressed on the cell intima,GSK3?,? were expressed in the cytoplasm and TCF-4 protein was highly expressed in the nucleus.2.WB results showed that the LRP5/6 protein was not expressed on the cell membrane in the ovarian tissue of mice.The results were not obviously changed after phosphorylation of LRP5/6.Further experiments were needed after antibody replacement.3.WB results showed that DVI3 and TCF-4 proteins were highly expressed in FVB mice,but it was low in ICR mouse ovarian cells.Theoretically,the molecule is very important and conserved,why there are differences between species remains to be further studied.4.The results indicate that Wnt signaling pathway plays an important role in the formation of mouse ovarian cells and follicles.It may stimulate the activation of DVI3 on the endometrium through the phosphorylation of LRP5/6 on the cell membrane,which further regulates the intracellular GSK3a,p and activates ?-catenin,and then regulate the TCF transcriptional regulatory factor in the nucleus,and start the gene transcription.Chapter 2:The regulation of Wnt/p-catenin signaling pathway on the Cumulus granulosa cells in PCOS patientsMethod:1.The ova in different stage and cumulus granulosa cells were collected in PCOS patients after stimulation by super long down-regulation protocol2.Preparation of cell crawling slices.3.Immunofluorescence assay LRP5/6,DVI3,GSK3?,?,CDH1 and CTNNB1 proteins of the cumulus granulosa cells.4.Western Blot was used to detect the expression of LRP5/6,DVI3,GSK3?,?,TCF-4,CDH1 and CTNNB1 proteins.Result:1.Immunofluorescence assay was used to detect the localization of protein in the cumulus granulosa cells.(1).Immunofluorescence assay was used to detect the localization of LRP5/6,DVI3,GSK3?,? proteins in granulosa cells,the LRP5/6 and DVI3 proteins were present on granulosa cell membrane,and the GSK3?,? protein was present in granulosa cytoplasm.(2).Immunofluorescence assay was used to detect the localization of CDH1 and CTNNB1 proteins in granulosa cells,CDH1 and CTNNB1 proteins were present in granulosa cytoplasm.2.Western Blot was used to detect the expression of proteins.(1).Western Blot was used to detect CDH1 and CTNNB1 protein of the cumulus granulosa cells,CDH1 and CTNNB1 protein were highly expressed in cumulus granulosa cells.The results showed that the content of CDH1 protein in MII group was 1.4093 ±0.2566,and it was 1.4124± 0.0575 in GV group,the content of CTNNB1 protein was 0.0595 ±0.0155 in group MII,and it was 0.0588 ±0.0078 in group GV,there was no significant difference in the expression of CDH1 and CTNNB1 proteins in granulosa cells of different maturity.(2).Western Blot was used to detect LRP5/6,GSK3?,?,DVI3 and TCF-4 protein in the cumulus granulosa cells,LRP5/6,GSK3?,?,DVI3,and TCF-4 protein in cumulus granulosa cells were not detected by WB assay.Conclusion:1.Immunofluorescence assay showed that the LRP5/6 and DVI3 proteins were present on granulosa cell membrane and the GSK3?,?,CDH1 and CTNNB1 proteins were present in granulosa cytoplasm.2.WB results showed that CDH1 and CTNNB1 proteins were highly expressed in cumulus granulosa cells,there was no significant difference in the expression of CDH1 and CTNNB1 proteins in granulosa cells of different maturity.However,the content of CDH1 protein decreased with the maturation of granulosa cells,and the content of CTNNB1 protein increased with the maturation of granulosa cells.LRP5/6,GSK3?,?,DVI3 and TCF-4 protein in cumulus granulosa cells were not detected by WB assay.Further experiments were needed to replace antibodies or Expand sample size,3.The results indicate that Wnt signaling pathway plays an important role in the development of follicles.It may stimulate the activation of DVI3 on the endometrium through the phosphorylation of LRP5/6 on the cell membrane,which further regulates the intracellular GSK3?,? and activates ?-catenin,and then regulate the TCF transcriptional regulatory factor in the nucleus,and start the gene transcription.PART ?:Genomic editing of Wnt/?-catenin signaling pathwayMethod:1.Construction of genomic editing vector for Wnt/?-catenin signaling pathway.2.Plasmid transfection.3.Direct microinjection of fluorescent expression vector.4.Fluorescent expression vector was transfected into granulosa cells.5.Editing of the human genome.Result:1.Construction and validation of FnCpfl vector:In order to study the role of Wnt/beta-catenin signaling pathway in the pathogenesis of PCOS,we designed the relevant CrRNA sequences for four corresponding genes.2.Plasmids were injected into abandoned fertilized eggs to express corresponding proteins.3.The transfection efficiency of human granulosa cells is low.4.The recombinant protein could be used to edit VEGFA gene after microinjection.Conclusion:1.A genomic editing vector for Wnt/beta-catenin signaling pathway was successfully constructed by designing relevant CrRNA sequences for four corresponding genes in Wnt pathway.2.The transfection efficiency of plasmid granule cells is low.It is necessary to optimize transfection conditions or to change lentivirus transduction.3.CRISPR/SaCas9 can edit genome of human fertilized eggs.