The Role And Mechanism Of Meiotic Genes MEIOSIN,KASH5 And BRCA2 In The Pathogenesis Of Premature Ovarian Insufficiency

Chapter Ⅰ Study on the role of MEIOSIN mutation in the pathogenesis of premature ovarian insufficiencyBackground:Premature ovarian insufficiency(POI)is a disorder wherein ovarian function ceases before age 40.Etiologic research is important for the early recognition and diagnosis of POI.The etiology of POI is complex and highly heterogeneous,including genetic,immunological,environmental and iatrogenic factors.Genetic defects have been implicated in 20-25%of POI patients in which search for novel causative genes has been a major focus.In recent years,several meiotic genes have been identified as causative genes of POI through whole exome sequencing(WES).The importance of meiotic genes for maintaining ovarian function has received wide attention.The meiosis initiation of oocytes is regulated by multiple signaling pathways,such as retinoic acid,bone morphogenetic protein,and Wnt/β-catenin pathways.Previous studies have uncovered that STRA8 and MEIOISN variants were associated with POI.However,the role of meiosis initiation genes in the pathogenesis of POI still needs further investigation.Objective:To elucidate the role and mechanism of meiosis initiation genes in the pathogenesis of POI.Methods:We screened bi-allelic variations of 13 genes involved in meiosis initiation in the in-house WES database,which were confirmed by Sanger sequencing.Protein structure prediction and dual-luciferase reporter assay were performed to verify the pathogenicity of the variation.To demonstrate the effect of the variant on oogenesis,we constructed a knock-in mice model with the homologous mutation and observed the reproductive phenotype of the point mutation mice.The role of the variant in meiosis was explored by immunofluorescence staining for SYCP1 and SYCP3.Further immunofluorescence staining was performed to detect the location of mutant protein and the cell cycle of meiosis.The mRNA levels of meiotic genes in the oocytes were measured by quantitative real-time PCR.Results:A homozygous variant in MEIOSIN(c.1735C>T,p.R579W)was identified in a patient with idiopathic POI,which localized to the HMG box,a DNA-binding motif.The variant site was conserved across species.The predicted protein structure of MEIOSIN-STRA8 suggested that the variant changed the surrounding structure and weakened the interaction between the proteins.The dual-luciferase reporter assay revealed that the mutant MEIOSIN exhibited diminished abilities to activate the transcription of downstream meiotic genes.The homozygous mutant mice with homologous Meiosin mutation were infertile with ovarian atrophy.Histological analysis of ovarian tissue revealed that the oocytes were reduced at 3 days postpartum(P3)and depletion at P21.The immunofluorescence staining for SYCP1 and SYCP3 suggested that the meiosis progress of mutant oocytes was arrested.Further immunofluorescence staining and quantitative real-time PCR confirmed that the mutant MEIOSIN localized in the cytoplasm,resulting in a decrease in mRNA levels of meiotic genes.Conclusion:In this study,we identified a pathogenic homozygous MEIOSIN variant in a POI patient.The in vitro experiments and point mutation mouse model confirmed that the variant affected the transcription of a set of meiotic genes and accelerated oocyte depletion.Our study firstly demonstrated that MEIOSIN is the causative gene of POI,elucidated the contribution of meiosis initiation genes in the pathogenesis of POI and emphasized the importance of proper meiosis initiation for maintaining ovarian function.Chapter Ⅱ Study on the role of KASH5 mutation in the pathogenesis of premature ovarian insufficiencyBackground:After meiosis initiation,oocytes undergo programmed DNA double-strand breaks(DSBs),and maintain genomic diversity and stability by synapsis and recombination between homologous chromosomes.In this process,the telomere-mediated rapid chromosome movement clusters chromosomes to one side of the nucleus,forming a meiotic "bouquet" to promote the recognition of homologous chromosomes.To power chromosome movements,KASH5 and SUN 1 form a LINC complex which links the dynein to telomeres.However,the role of a defective LINC complex in the pathogenesis of POI was unknown.Objective:To elucidate the role of KASH5 mutation in the pathogenesis of POI.Methods:We performed WES in a consanguineous pedigree with two POI patients.Candidate variants were screened out and validated by Sanger sequencing.The location of mutant KASH5 was illustrated by immunofluorescence staining,and we performed co-immunoprecipitation to examine whether mutant KASH5 interacted with SUN 1.To demonstrate the effect of the variant on ovarian function and oogenesis,we observed the reproductive phenotype of the Kash5 C-terminal deleted mice.The effect of the variant on meiosis was confirmed by oocyte spreads and immunofluorescence staining for SYCP1 and SYCP3.Results:A homozygous splicing site variant in KASH5(c.747G>A,p.Ala249Ala)was identified.Minigene assay demonstrated that the variant affected the RNA splicing of KASH5,resulting in a C-terminal truncated protein.We overexpressed wild-type and mutant KASH5 plasmids and SUN1 plasmids in HeLa cells,and found that the variant disturbed the nuclear membrane localization of KASH5.Co-immunoprecipitation showed that truncated KASH5 protein lost the interaction with SUN1.Further studies of KASH5 C-terminal deleted mice revealed that homozygous mutant mice were infertile with ovarian atrophy.Histological analysis of ovarian tissue revealed that the oocytes were reduced at P3 and depletion at P21.The immunofluorescence staining for SYCP1 and SYCP3 showed that the meiosis progress of mutant oocytes was arrested at a zygotene-like stage,suggesting that the pairing of homologous chromosomes was interrupted.Conclusion:In this study,we identified a homozygous splicing site variant of KASH5 in a POI pedigree.The in vitro and in vivo experiments suggested that the variant disturbed meiotic homolog pairing and accelerated oocyte depletion.Our study firstly demonstrated that disorganized LINC complex was involved in the pathogenesis of POI,suggesting the essential roles of telomere-mediated chromosomal movement-related genes in ovarian function maintenance.Chapter Ⅲ Study on the mechanism of BRCA2 mutation in the pathogenesis of premature ovarian insufficiencyBackground:During the process of synapsis,genetic material exchanges between homologous chromosomes,which is the core molecular event of meiotic prophase I.BRCA2 is widely recognized as a central regulator of homologous recombination(HR).In spermatocytes,BRCA2 recruits RAD51 and DMC1 to DSBs.However,the role of BRCA2 in the meiotic HR of oocytes is still unclear.In recent years,biallelic variants of BRCA2 were identified in familial and sporadic POI cases,while phenotype distinctions existed among carriers of different variants.The mechanism of BRCA2 variants in the pathogenesis of POI needs further investigation.Objective:To explore the role of BRCA2 in meiosis of oocytes and elucidate the mechanism of BRCA2 variation in the pathogenesis of POI.Methods:We constructed knock-in mouse models mimicking the compound heterozygous BRCA2 variants identified in POI patients and observed the reproductive phenotype.The H&E staining,immunofluorescence staining for DDX4 and Cleaved-PARP1 were performed to demonstrate the effect of the variant on oogenesis.Oocyte spreads and immunofluorescence staining for various markers were performed to clarify the effect of Brca2 variants on meiosis and the role of BRCA2 in meiotic HR.Results:The adult Brca2c.68-1G>/c.4384-4394del mice were infertile with ovarian atrophy.Histological analysis of ovarian tissue revealed that the oocytes were significantly reduced at P21.The immunofluorescence staining for DDX4 and Cleaved-PARP1 revealed that the oocytes underwent massive apoptosis after birth.The progression of meiotic prophase I was examined by immunofluorescence of oocytes against SYCP1 and SYCP3,which showed synaptic abnormalities in mutant oocytes.Immunofluorescence of oocytes against yH2AX showed that oocytes of mutant ovaries revealed persistent staining of yH2AX at the pachytene-like and diplotene-like stages,indicating meiotic HR defects in the mutant oocytes.HR markers RPA,RAD51 and DMC1 were analyzed by immunofluorescence staining on oocyte spreads.The RPA foci showed almost comparable spatiotemporal distribution between wild-type and mutant oocytes,while the number of RAD51 and DMC1 foci were significantly reduced in leptotene and zygotene oocytes from mutant mice,suggesting that the Brca2 variants impaired the recombinase recruitment to DSBs in the meiotic HR of oocytes.Conclusion:In this study,we confirmed that the BRCA2 variants c.68-1G>C plus c.4440T>G(p.Y1480X)induced oocyte exhaustion by synaptic abnormalities and delayed DSB repair due to impaired meiotic HR.Our findings also demonstrated in mice that BRCA2 recruited recombinases in the meiotic HR of oocytes.