| In recent years,it has been reported that a number of pathogenic genes led to human oocyte maturation disorder,fertilization disorder,early embryonic development block and other types of infertility,but there are few reports of pathogenic genes in patients with fertilized egg mitosis disorder(cleavage disorder).There is no effective treatment.In order to identify the pathogenic gene of cleavage disorder and analyze its pathogenic mechanism,in the first chapter of this study,we collected infertile patients and families with cleavage disorder for full exon sequencing.After screening and verification of a series of sites,we identified the pathogenic mutation of CHK1 gene.In-depth mechanism studies have shown that these mutations may affect the structure of CHK1 protein,change the nuclear and cytoplasmic location of protein,lead to the increase of CHK1 kinase activity,and cause cell cycle arrest through CDC25C/CDK1 pathway,thus making the patient show cleavage disorder.The diagnosis of this cleavage disorder patient provides a new molecular target.Considering that the patients with CHK1 pathogenic mutation are accompanied by obvious prokaryotic membrane rupture disorder,in order to further explore its possible mechanism,in the second chapter of this study,we found that the F-actin cytoskeleton in the CHK1 mutant fertilized egg was destroyed through the nucleocytoplasmic exchange of the fertilized egg and the single cell transcriptome sequencing of the fertilized egg of the patient.The mutant CHK1 protein may affect the enzyme activity of the latter by changing the interaction with MICAL3 protein,which leads to the depolymerization of F-actin.It affects the rupture of pronuclear membrane,which provides a specific mechanism for the regulation of prokaryotic membrane rupture of mammalian fertilized eggs.After clarifying the pathogenic mechanism,in order to explore the possible treatment strategies for patients with this kind of cleavage disorder,in the third chapter of this study,PF477736,an inhibitor of CHK1 protein,was used to inhibit the increased enzyme activity of CHK1 mutant protein.The fertilized eggs carrying mutants in mice were treated with PF477736 to develop into blastocysts and produce healthy offspring.Patients’ mutant fertilized eggs can also obtain high quality and developmental potential blastocysts after PF477736 treatment.This provides an effective intervention for the treatment of infertile patients with CHK1 gene mutation.Chapter 1 The pathogenic mechanism on zygote cleavage failure caused by CHK1 mutationObjectiveA large proportion of embryos obtained by assisted reproductive technology are blocked in the early embryonic stage,in which the block of fertilized eggs is called "zygote cleavage failure".Patients with zygote cleavage failure can not obtain transferable embryos after several assisted reproductive technology cycles,but its pathogenic etiology is unclear.The purpose of this chapter is to deeply study its genetic etiology and analyze the specific pathogenic mechanism.MethodsIn order to explore the genetic etiology of zygote cleavage failure,we first collected the peripheral blood from 29 patients with zygote cleavage failure characterized by obvious pronuclei,to perform whole exome sequencing.Then the possible pathogenic variants were screened and filtered by a series of bioinformatics analysis and verified in 300 normal controls.Secondly,in order to clarify the pathogenicity of the finally screened CHK1 mutations,the zygote cleavage disorder phenotype of patients was reproduced by microinjection of CHK1 cRNA into mouse fertilized eggs.In addition,to explore the effect of CHK1 mutation on protein function,we constructed the structural model of mutant protein by homology-modeling and detected its localization in cells by immunofluorescence.Next,in order to further explore the mechanism on how CHK1 mutations affect zygote cleavage,we detected the kinase activity of mutant protein by CHK1 kinase activity assay,and detected the phosphorylation level of their downstream factors,CDC25C and CDK1,by Western Blot.Finally,in order to verify the essential role of CDC25C/CDK1 pathway in zygote cleavage failure induced by CHK1 mutations,we mutated the key phosphorylation sites of CDC25C and CDK1,and then overexpressed the mutated CDC25C and CDK1 in mouse fertilized eggs by microinjection together with CHK1 mutation to observe its effect on the division of fertilized eggs.Results1.Identification of CHK1 pathogenic mutations in patients with zygote cleavage failureWe first focused on a family in which the proband could not obtain transferable embryos after three cycles of assisted reproductive treatment in our hospital;when the normal control fertilized eggs developed to the 8-cell stage,eggs from the proband were blocked with clear pronuclei.The patient’s sister and an aunt also had a similar history of infertility,showing an autosomal dominant inheritance pattern.We then sequenced the proband and two female controls,her mother and a health aunt in the family.After a series of rigorous site screening and verification,we finally mapped a mutation in the CHK1 gene(p.R379Q).Next,we found three more CHK1 mutations in other 27 patients with zygote arrest and pronuclear rupture disorder.and separately traced to three families,with a mutation rate of 24%.The four inherited or de novo CHK1 mutations,which had not been reported in the public database,were predicted to be highly damaging by softwares(SIFT、Polyphen、MutationTaster),the ACMG scores indicated these mutations to be likely pathogenic,and the corresponding amino acid residues were highly conserved among species.2.Mimic the CHK1 mutation phenotype in mouse fertilized eggsWestern blotting and quantitative PCR of oocytes and preimplantation embryos show that CHK1 is highly expressed in zygote and 2-cell stage,suggesting that CHK1 may play an important role in the fertilized egg.The cleavage rates of mouse fertilized eggs overexpressed with mutant CHK1 were significantly lower than that of fertilized eggs overexpressing wild type CHK1,especially in the truncated mutation group(p.F441fs*16).Besides,when the fertilized eggs expressing wild type CHK1 developed to 2-cell stage.the fertilized eggs from mutant groups still did not divide and clear pronuclei could be seen in most of the arrested eggs,which was consistent with the phenotype of patients with CHK1 mutations.3.Altered conformation and nucleo-plasmic localization of mutant CHK1 proteinThe homology-modeling of mutant CHK1 protein showed that missense mutations R379Q and R442Q changed their hydrogen bond with surrounding amino acid residues,and the mutations also affected surface potential of CHK1 protein due to the change of amino acid properties.As for the nucleo-plasmic localization,wild-type CHK1 protein was mainly localized in the nucleus,while the signals in cytoplasm of mutant CHK1 were enhanced with the truncated mutation(p.F441fs*16)mainly located in the cytoplasm.The four pathogenic CHK1 mutations are located in the conserved motif of CHK1 C-terminal,corresponding to nuclear export signal and nuclear localization signal.After the inhibitor,Leptomycin B,was used to suppress the nuclear export signal,all missense mutations restored nuclear localization except for the truncated mutation,indicating that the changed localiztion of these mutations were due to the destruction of nuclear export signal or nuclear localization signal.The changes in nuclear and cytoplasmic localization of CHK1 mutation may affect its biological function.4.CHK1 mutations result in zygote cleavage failure through CDC25C/CDK1 pathwayCHK1 exhibited increased kinase activity in all mutant groups,and the mutant CHK1 could increase the expression of inhibitory phosphorylated CDC25C(S216)and CDK1(T14 and Y15).As expected,the cleavage rate of mutant fertilized eggs(p.F441fs*16)increased significantly after the key phosphorylation sites of CDC25C and CDK1 were mutated to be inactivate.To sum up,the results suggest that the activity of CHK1 is increased after mutation,which leads to cleavage disorder through CDC25C/CDK1 pathway.ConclusionBy collecting blood samples from patients with zygote cleavage failure for whole exome sequencing,we found four heterozygous mutations of CHK1 gene in four independent families.The overexpression of CHK1 mutation in mouse fertilized eggs could mimic the phenotype of zygote cleavage failure with unruptured pronuclear member.In-depth mechanism study inferred that these four CHK1 mutations changed the protein conformation,affected the necleocytoplasmic localization,increased the kinase activity and caused cleavage disorder through inhibitory phosphorylation of downstream CDC25C/CDK1 pathway.The study in this chapter provides a new target for the diagnosis of infertile patients with zygote cleavage failure and demonstrates its specific pathogenic mechanism.Chapter 2 The mechanism on the failure of pronuclear envelope breakdown affected by CHK1 mutationObjectiveThe rupture of pronuclear membrane in mammalian fertilized eggs is an important guarantee for the successful initiation of the first mitosis,but its regulatory mechanism is still unclear.The blocked fertilized eggs from patients with CHK1 mutation are accompanied by obvious pronuclear envelope.The purpose of this chapter is to analyze the specific molecular mechanism on how CHK1 regulating the rupture of pronuclear envelope of fertilized eggs.MethodsIn view of the enhanced cytoplasmic localization of the mutant CHK1 protein,we first used the pre-pronuclear transfer technique to carry out nuclear-cytoplasmic exchange between the patient’s mutant fertilized egg and the normal donor fertilized egg,in order to detect whether the mutation mainly affects the biological process in the cytoplasm of fertilized eggs.Next,to study the key processes or pathways affected by CHK1 mutation,we collected fertilized eggs donated by patients for single cell transcriptome sequencing and discovered downregulation of cytoskeleton components.We then focused on F-actin,a key cytoskeleton component,and utilised mouse fertilized eggs with overexpressed mutations to verify its regulatory effect on pronuclear envelope breakdown.Afterwards,to explore how CHK1 mutations regulate depolymerization of F-actin in the fertilized egg,we went to identify the essential substrate of CHK1 by means of co-immunoprecipitation for 6000 mouse fertilized eggs and further validated the interaction by proximity ligation assay in human embryos and coimmunoprecipitation in HEK-293 cells.By fluorescence resonance energy transfer,protein modeling and docking,and co-immunoprecipitation,we detected the conformation change of CHK1 mutation and interaction difference between CHK1 mutations and MICAL3.With the purpose to assay the enzyme activity of MICAL3,we subsequently performed hydrogen peroxide production detection and F-actin polymerization detection.Finally,MICAL3’s activity was inhibited by EGCG in fertilized eggs carrying CHK1 mutation to rescue the reduced zygote cleavage rate and further verify the vital role of MICAL3 in pronuclear envelope breakdown,Results1.Patient’s zygote developed into blastocyst afer replacement of cytoplasmThe cytoplasm exchange between the fertilized egg of the patient and the normal donor was carried out by pre-pronuclear transfer technique,and the patient’s eggs could successfully develop to blastocyst after cytoplasmic replacement.Further research demonstrated an embryonic stem cell line,derived from the reconstructed blastcyst,was established and was validated to be with pluripotency and complete genomic ploidy.These results suggest that the mutation mainly influence the cytoplasm of patient’s fertilized eggs.2.CHK1 mutation may affect pronuclear envelope breakdown by disturbing F-actinBy single-cell transcriptome for the patient’s fertilized eggs,we discovered downregulated cytoskeleton components in mutated eggs.In view of the fact that the pronuclear membrane is also not diasppear after overexpression of CHK1 mutation in mouse fertilized eggs,which is consistent with the patients’ phenotype.We then observed the expression of F-actin in mutant fertilized eggs and found the expression of F-actin in mutant fertilized eggs decreased significantly.Further assembly inhibition of F-actin or microtubule could block the development of fertilized eggs,but only F-actin inhibition hindered the rupture of pronuclear envelope in fertilized eggs,indicating the critical role of F-actin in regulating pronuclear envelope breakdown.3.The interaction between CHK1 mutation and MICAL3 is enhanced6000 mouse fertilized eggs collected by in vitro fertilization were used for coimmmunoprecipatation together with mass spectrum to identify the special substrate of CHK1,MICAL3,who mainly localized in the cytoplasm both in oocytes and HEK-293 cells.Then their interaction was identified by immunoprecipitation in HEK-293 cells and by proximity ligation assay both in human embryos and HEK-293 cells.Given that the "self-inhibitory"conformation of CHK1 mutations were altered,verified by fluorescence resonance energy transfer experiments,we then checked the interaction between CHK1 mutants and MICAL3 and found their enhanced interaction,implied by protein-protein docking and coimmunoprecipitation.4.Mutant CHK1 increased MICAL3’ activity to accelerate F-actin depolymerizationMICAL protein family belongs to monooxygenase and can oxidize actin monomer to mediate F-actin depolymerization.Our results show that mutant CHK1 protein was able to enhance MICAL3 activity and promote F-actin depolymerization.In addition,the application of MICAL3 inhibitor EGCG to inhibit its monooxygenase activity could make the mutant fertilized eggs recover cleavage and develop to blastocyst.ConclusionThe blastocysts with normal development potential can be obtained by transferring the pronucleus of fertilized eggs from patients with CHK1 mutation into the cytoplasm of normal enucleated fertilized eggs,suggesting that the mutation may seriously destroy the biological events in the cytoplasm.Further studies have shown that CHK1 mutation is likely to affect the process of pronuclrear membrane breakdown by destroying the F-actin network in fertilized eggs,which might result from increased activity of MICAL3.The series studies in this chapter provide a theoretical basis for the regulation mechanism of the important biological process,pronuclear envelope breakdown,in mammalian fertilized eggs.Chapter 3 Application of CHK1 inhibitor to explore intervention strategy for patients with zygote cleavage failureObjectiveAt present,there is no effective treatment for patients with zygote cleavage failure.After verifying its genetic etiology and specific pathogenesis,in-depth exploration of its potential treatment strategy is expected to benefit infertile patients with CHK1 mutation.The purpose of this chapter is to explore a potential intervention measure for patients with CHK1 mutation and to verify its efficacy and safety.MethodsIn order to test whether the CHK1 inhibitor PF477736 could make the fertilized eggs carrying CHK1 mutation divide normally,we first treated the mouse fertilized eggs cultured in vitro with PF477736 under reported concentration and observed their cleavage.Then to explore the optimal concentration,different concentrations of PF477736(0/1/10/100nM)was applied to observe the blastocyst development of mutant fertilized eggs.What’s more,with the optimal concentration(10nM),we preformed embryo transfer to further detect its effectiveness.As to rescue effect of the inhibitor on patients’ fertilized eggs,we first treated patient’s fertilized eggs,which had been cultured in vitro for three days but still did not divide,with PF477736.Then patients’ fresh fertilized eggs were treated with PF477736 to measure its ability for developing into blastocysts.Finally,in order to access the developmental potential of blastocysts treated with PF477736,we constructed and estimated the derived embryonic stem cell lines by immunofluorescence,copy number variant analysis and genotype identification.Results1.CHK1 inhibitor rescued mouse zygote cleavage failure and help to obtain offspingIn terms of the increased kinase activity of the CHK1 mutations,we considered to employ CHK1 inhibitor PF477736 for carrying out rescue experiment.We first found that PF477736 could reduce the level of phosphorylated CDC25C/CDK1 in each mutant group.Preliminary tests showed that PF477736 could significantly increase the cleavage rate of mutant fertilized eggs.Then,we used mouse fertilized eggs overexpressing CHK1 mutation to explore the optimal concentration of PF477736(10nM).At this concentration,the mouse mutant fertilized eggs could develop into blastocysts with complete genomic ploidy and produce healthy offspring.2.CHK1 inhibitor made patient’s zygote develop into high-quality blastocystTo our surprise,the retarded fertilized eggs from patient’s were able to resume cleavage after PF477736 treatment.Therefore,we applied PF477736 to the fresh fertilized eggs donated by the patients and found 2 out of 5 fertilized eggs developed into high-quality blastocysts.To examine the developmental potential of these blastocysts,we also established embryonic stem cell lines and discovered they held pluripotency and complete genomic ploidy,which provided an effective method for the accurate treatment of patients carrying CHK1 mutation.ConclusionApplication of CHK1 inhibitor could effectively rescue the zygote arrest phenotype of fertilized eggs in both mice and patients caused by CHK1 mutation,and produce healthy mouse offspring or high-quanlity human blastocyst.Researches in this chapter thus provide a potential intervention for patients with zygote cleavage failure. |
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