| Human infertility is a rising issue affecting 10-15%reproductive-aged couples worldwide.In recent years,the incidence of infertility is increasing with the increase of social pressure,the delay of childbearing age and the aggravation of environmental pollution.Assisted reproductive technology（ART）has helped millions of patients to conceive a child.However,about 10%of embryos achieved by ART are arrested at the early cleavage stage,which is clinically diagnosed as early embryonic arrest（EE A）.EE A is an important cause of recurrent ART failure;however,there is lacking predicting indicators for early diagnosis in clinics.Therefore,it is of great significance to explore the causative factors and reveal the mechanisms of EEA.Successful fertilization and embryogenesis require oocyte maturation.Multiple biological events occur during oocyte meiotic maturation,fertilization and early embryonic development,such as meiosis,maternal RNA clearance,and zygotic genome activation（ZGA）.The abnormality in either process leads to EEA.Recently,with the development of whole-exome sequencing（WES）,several maternal-effect genes have been found to be responsible for EEA,such as MOS,NLRP2,NLRP5,TLE6,KHDC3L,PADI6 and TUBB8.However,the underlying genetic factors remains largely elusive.NLRPs（Nucleotide-binding oligomerization domain,Leucine rich Repeat and Pyrin domain containing Proteins）,as pattern recognition receptors,are well recognized for their important roles in both mammalian innate immune system and reproductive system.NLRP2 and NLRP5 that have been found involved in EEA belong to the NLRPs family,suggesting that NLRPs play important roles in early embryonic development.As a gonad-specific expression member of NLRPs,NLRP14 functions in spermatogenesis and NLRP14 mutations have been found in men with spermatogenic failure.Recent studies have also reported that Nlrp14 SiRNA-injected zygotes showed embryonic development arrest at the 1～8-cell stage,but the mechanism was still unclear.The exact role and the molecular mechanisms of NLRP14 in oocyte meiotic maturation and early embryonic development remain unclear.Moreover,NLRP14 has not been found to be associated with female infertility.Objective:To elucidate the role and mechanism of NLRP14 in oocyte maturation and early embryonic development,and explore the pathogenicity of NLRP14 variants in patients with EEA.Methods:In Chapter Ⅰ,to investigate the role of NLRP14 in oocyte maturation and early embryonic development,the localization and expression of NLRP14 in different tissues,oocytes and embryos within different developmental stages were revealed by WB,qPCR and IF of wild-type female mice.By the CRISPR/Cas9 system we generated Nlrp14 knockout mice.The effect of Nlrp14 on female fertility was investigated by counting the litter size of Nlrp14-/-mice.The role of Nlrp14 in oocyte meiotic maturation and early embryonic development was explored by IVM and IVF.The oocyte quality makers,such as spindle,chromosome,mitochondria,cytoplasmic lattices（CPLs）and chromatin were observed by IF.The NLRP14 interacting proteins were investigated by IP-MS.The proteins expression was explored by proteomics analysis.To explore the role in RNA transcription,clearance and zygotic genome activation（ZGA）,oocytes at GV and MII stages,zygotes（Nlrp14♀-/♂+）,and two-cell（Nlrp14♀-/♂+）embryos were collected for RNA-seq analyses.In Chapter Ⅱ,to determine whether NLRP14 variants are associated with EEA,we screened the bi-allelic variants of NLRP14 in WES database including 606 patients with EEA.To elucidate the pathogenesis of NLRP14 mutations,we compared the interaction between UHRF1 and wild-type or mutant NLRP14 protein by overexpressed wild-type or mutant NLRP14 and UHRF1 in HEK293 cells.Moreover,we evaluate their effects on UHRF1 expression through oocytes microinjection with wild-type or mutant NLRP14 mRNA.Results:1.The role and mechanism of NLRP14 in oocyte meiotic maturation and early embryonic development1.1 The localization and expression of NLRP14 during oocyte meiotic maturation and early embryonic developmentThe mRNA and protein level of different organs/tissues from wild-type female mice showed that NLRP 14 is preferentially expressed in ovary but not in other tissues.The mRNA expression in the ovaries,oocytes and early embryos from wild-type female mice revealed that Nlrp14 is preferentially expressed in oocytes and quickly disappeared after fertilization.Immunofluorescence staining of ovary sections,oocytes and early embryos from wild-type female mice revealed that NLRP 14 protein was expressed in as early as the primordial follicle stage and maintained till morulae stage.NLRP 14 mainly localized in the subcortex of oocytes and early embryos.Besides,the qPCR and RNA-seq of human samples showed that NLRP14 is preferentially expressed in oocytes and early embryos.In summary,the specific expression pattern in oocytes and early embryos suggested that NLRP14 may be a maternal-effect gene and play a critical role during oogenesis and early embryogenesis.1.2 The role of NLRP14 in oocyte maturation and early embryonic developmentWe generated a Nlrp14 knockout mouse strain and found that Nlrp14-/-female mice were infertile.Results of in vitro fertilization showed that Nlrp14-/-oocytes derived embryos arrested at the 1-cell or 2-cell stage and none developed beyond the 2-cell stage.The oocytes in vitro maturation revealed that the germinal vesicle breakdown（GVBD）process was normal.However,polar body-1（PB1）emission rate was lower in Nlrp14-/-oocytes than that in wildtype oocytes.Similarly,the frequency in PB1 emission rate was reduced in Nlrp14-null oocytes in vivo.These results suggested that Nlrp14-/-mice showed EEA due to oocyte meiotic defects.1.3 The mechanism of Nlrp14 deletion affects oocyte meiotic maturation and early embryonic developmentWe found that nearly 50%of Nlrp14-/-MII oocytes had obvious abnormalities in spindle organization,and 85%exhibited defects in chromosome congression,alignment and segregation.Besides,although with normal morphology in GV oocytes,we found impaired mitochondria distribution,lack of CPLs,and decreased surrounded nucleolus（SN）ratio in Nlrp14-/-oocytes.Taken together,these results indicated that Nlrp14 deletion reduces oocytes quality and impairs oocyte meiotic maturation.To investigate how Nlrp14 loss affects oocyte meiotic maturation and early embryos development,IP-MS was used to identify potential NLRP14 interacting proteins and mainly included proteins involving in spindle assembly-associated microtubule,subcortical maternal complex（SCMC）,methylation and transcription.To further investigate the molecular mechanisms,we performed proteomics analysis and found downregulated proteins enriched in microtubules and methylation.We confirmed the decreased expression of microtubule proteins（TUBB3,TUBA1,TUBA4A）by western blot in the Nlrp14-/-oocytes,which could explain the spindle assembly abnormalities phenotype.IP-MS showed SCMC proteins are NLRP14 interacting proteins.However,Nlrp14 deletion is not essential for the assembly of SCMC complex and NLRP14 does not act through the SCMC functional module.IP-MS and proteomics analysis indicated that UHRF1 is the protein that declines the most in the Nlrp14-/-oocytes group and is also obviously enriched by IP.Through western blot and immunofluorescent staining,we confirmed that the expression of UHRF1 protein was significantly reduced in the Nlrp14-/-oocytes.Co-IP showed that NLRP14 could specifically precipitate UHRF1,as well as UHRF1 could specifically precipitate NLRP14 in wild-type mouse oocytes lysates.Treatment of Nlrp14-/-GV oocytes with the proteasome inhibitor MG132 increased levels of UHRF1,which indicated that NLRP14 can bind with UHRF1 and protect it from proteasome-dependent degradation.To investigate the roles of NLRP14 in RNA transcription,degradation and ZGA,we collected oocytes at GV and MII stages,zygotes（Nlrp14♀-/♂+）,and two-cell（Nlrp14♀-/♂+）embryos of both genotypes（wild-type and Nlrp14-/-）for transcriptome sequencing.Gene ontology（GO）analysis revealed that differential expression genes were involved in cytoskeleton and spindle assembly,chromosome organization and segregation,mRNA processing and transcription,mitochondrial distribution and cell cycle,in consistent with the phenotype.In addition,the analysis showed that Nlrp14 deletion resulted in Z-decay and ZGA defects,which might lead to meiotic defects and early embryonic arrest.2.The role of NLRP14 mutations in the pathogenesis of EEA2.1 NLRP14 compound heterozygous variants were identified in EEAIn WES database of 606 patients with EEA,we identified a compound heterozygous variant,composed of a frameshift variant c.12821283delTG（p.Cys428Profs*28）and a deletion-insertion variant c.2660delinsGCTA（p.Leu887delinsArgTyr）（NM176822.4）in a infertile woman.The recessive inheritance pattern was confirmed via Sanger sequencing.The variant c.12821283delTG had a low frequency of 0.001554 and the variant c.2660delinsGCTA has not been reported in the gnomAD exome database.2.2 The function of mutant NLRP14 proteinsIn Chapter I,we found that NLRP14 could interact with UHRF1 in oocytes and the UHRF1 expression was decreased in Nlrp14 deficient oocytes.Therefore,we further assessed the function of the two NLRP14 variants on UHRF1.Co-IP was performed in HEK293 cells transfected with wild-type or mutant GFP-NLRP14 and MYC-UHRF1.The results showed that the truncated protein p.Cys428Profs*28 destroyed the interaction with UHRF1 and the mutant protein p.Leu887delinsArgTyr had significantly weaker binding capacity with UHRF1.Besides,microinjection with wild-type or mutant human NLRP14 mRNA were performed in mice oocytes to determine their effects on UHRF1 expression.Results showed that two mutant NLRP14 groups expressed much lower levels of UHRF1 than the wild-type group in oocytes.These results suggested that mutations in NLRP14 affected the interaction with UHRF1 and resulted in downregulation of UHRF1.Conclusion:Nlrp14 deletion in mice resulted in reduced oocytes quality,downregulation of microtubule proteins and UHRF1,and defects in mRNA Z-decay and ZGA,which contributed to oocytes meiotic defects and EEA and infertility in female mice.Besides,we identified and verified the pathogenic NLRP14 variants in EEA patients,which may facilitate the genetic counselling for EEA patients. |
