Zp1 Mutation Affects Oocyte Energy Metabolism And Leads To Oocyte Apoptosis

Background:Zona pellucida（ZP）is a transparent glycoprotein structure surrounding the oocyte.ZP begins to form with the activation of primordial follicles and accompanies the whole process of oocyte development and maturation.Human and rat ZP are composed of 4glycoproteins（ZP1-4）,and mice are composed of ZP1-3.ZP plays an important role in oogenesis,fertilization,and preimplantation development.Previous study reported the first case of Zp1 mutation causing human ZP deletion and infertility.Since the phenotype of model mice were different from human Zp1 mutation,rat model was constructed to study the mechanism of Zp1 mutation.On the basis of ZP and transzonal projections（TZPs）deletion and partial oocyte apoptosis,we propose to further study the changes of ovarian gene regulation in Zp1mutant rat.Objective:The purpose of this study was to explore the effects of Zp1 mutation leading to ZP and TZP abnormalities on the ovarian gene regulatory network,and provide molecular basis for oocyte development disorder by comparing the Zp1^WT/WTand Zp1^MT/MTrat ovarian transcriptomic sequencing analysis.Methods:1.Ovarian transcriptome sequencing in wild-type and Zp1^MT/MTrats:Taking Zp1^MT/MTrats as research object（n=5）,and wild-type rats as control group（n=5）.The RNA was extracted from the ovary and sequenced by BGIseq-500 platform.2.Ovarian sequencing data analysis in wild-type and Zp1^MT/MTrats:（1）Quality control and filtration of sequencing data;（2）Testing the correlation between the two groups;（3）Identification of differentially expressed genes（DEG）between the two groups:P value<0.05&Fold Change≥2;（3）Functional enrichment analysis of DEG:DAVID database;（4）miRNA prediction of DEG:Webgestalt and Cytoscape.3.Screening and verification of data analysis results in two groups:（1）DEGs in energy metabolic pathways were randomly selected and the significance of the pathway was verified by ordinary qRT-PCR;（2）Hypersensitive qRT-PCR and immunofluorescence were used to detect the expression of Pkm or Pdk4,which related to pyruvate production or decomposition;（3）DCFH-DA fluorescence probe was used to detect the level of reactive oxygen species（ROS）;（4）The expression of ROS target gene p53 and the activation level of downstream apoptotic protein caspase3 were detected by immunofluorescence.Results:1.Data analysis results:（1）A total of 20,001 genes were detected,including 293 DEGs;（2）The biological processes of GO enrichment analysis includes immune response and programmed cell death;cellular components includes extracellular matrix and extracellular domain;molecular functions includes RNA binding and G protein-coupled receptor activity;（3）KEGG enrichment analysis includes PI3K signaling pathway,Chemokine signaling pathway,cAMP signaling pathway and Energy metabolism pathway;（4）miRNA prediction of DEG showed that miRNA34A/C,miRNA181A/B/C,miRNA449 and miRNA214 had the largest number of target genes.2.Experimental verification results:（1）The expression trends of DEGs related to energy metabolism were consistent with the sequencing data;（2）There was no difference in the expression of Pkm in cumulus cells,while the expression of Pdk4 in Zp1^MT/MToocytes was significantly decreased;The level of ROS,the expression of ROS target gene p53,and the activation level of p53 downstream protein caspase3 were significantly increased in Zp1^MT/MToocytes.Conclusions:On the basis of ZP-deficient（deletion）in Zp1^MT/MTrat,this study found that 1.Zp1 mutation/ZP-deficient may affect oocyte maturation and development by affecting redox,cell migration,immunoreaction and energy metabolism;2.It was found that ZP deletion accompanied by Zp1mutation can lead to abnormal energy metabolism in oocytes,up-regulate the expression of reactive oxygen species target gene p53 and activate caspase3,resulting in oocyte apoptosis and infertility in female rats.