Zp2 Mutation Affects The Transduction Of TGF-β Signaling Pathway And Causes Oocyte Apoptosis

Background:The zona pellucida（Zona Pellucida,ZP）is an extracellular matrix surrounding mammalian oocytes,fertilized eggs and preimplantation embryos,which plays an important role in oogenesis,fertilization and maintenance of early embryonic development.Mammalian egg ZP contains multiple zona pellucida glycoproteins（ZPGs）,such as ZP1-3 in mice,ZP1-4 in human and rat.Mutations in the ZP genes caused human infertility for various causes of egg maturation and/or fertilization disorders,empty follicles and so on.The previous study of our research group found that homozygous mutations in rat Zp2led to ZP defects,granulosa cell translocation,oocyte apoptosis and female infertility.Transzonal projections that“cross”the ZP are formed between granulosa cells and oocytes,supporting the intercellular information and material exchange during egg maturation and development.Whether and how ZP defects affect oocyte-granulosa cells interactions remain unclear.Objective:In this study,wild-type(Zp2^wt/wt)rat was used as control and ZP-absent(Zp2^mut/mut)rat was used as research objects to explore the molecular mechanism of ZP defects affecting oocyte development and maturation from the level of oocyte transcriptome.Methods:1.Oocytes preparation and single cell RNA sequencingOocytes of Zp2^wt/wtand Zp2^mut/mutrat for single cell RNA sequencing（sc RNA-seq）were prepared by superovulation technique.We collected 4 oocytes from each group(Zp2^mut/mut:ZP-absent oocytes).Sequencing was performed by Smart-seq2.2.scRNA-seq data analysis of Zp2^wt/wtand Zp2^mut/mutoocytes（1）Oocytes sequencing data quality control.（2）Oocytes correlation analysis:Pearson algorithm.（3）Oocytes clustering:Facto Mine R and factoextra packages in R.（4）Selection of differentially expressed genes（DEGs）and heatmap drawing:DEseq2 and pheatmap packages in R.（5）GO and KEGG pathway enrichment analysis:DAVID.（6）mi RNA prediction of up-regulated DEGs:Web Gestalt and Cytoscape.3.Screening and validation of data resultsValidation of sequencing results:We selected 4 DEGs randomly,and real-time Quantitative Polymerase Chain Reaction（q RT-PCR）was used to verify the reliability of sequencing results.According to the previous data analysis results,the TGF-βsignaling pathway was selected for verification:（1）The expression levels of key receptor（Acvr2b）and ligands（Activin A and GDF8）in this pathway were verified by q RT-PCR and immunofluorescence.（2）The phosphorylation levels of the transduction proteins（Smad2 and p38MAPK）in this pathway were verified by immunofluorescence.（3）The expression level of the downstream gene Bcl2 in this pathway was verified by q RT-PCR and immunofluorescence.（4）The activation level of apoptosis protein caspase3 was verified by immunohistochemistry.Results:1.scRNA-seq data analysis of Zp2^wt/wtand Zp2^mut/mutoocytes（1）A total of 17,170 genes were detected.A total of 474 DEGs were selected.（2）The GO enrichment analysis showed that the biological processes were mainly involved in transcription,regulation and so on.The cellular components were mainly involved in exosomes,extracellular matrix and so on.The molecular functions were mainly involved in protein binding and DNA binding.（3）KEGG enrichment pathways mainly included protein processing in endoplasmic reticulum,oxidative phosphorylation,TGF-βsignaling pathway,TNF signaling pathway and so on.（4）The mi RNA prediction of up-regulated DEGs:The mi RNAs with the highest degree of connection included mi RNA29A,mi RNA29B and so on.2.Experimental verification results（1）The qRT-PCR results confirmed that the expression levels of 4DEGs randomly selected were consistent with the sequencing results.（2）In Zp2^mut/mutgroup of oocytes,the expression level of the receptor Acvr2b was significantly decreased.The expression levels of its ligands in granulosa cells were not significantly different.The phosphorylation levels of Smad2 and p38MAPK were decreased.The expression level of Bcl2 was significantly decreased and the apoptosis protein caspase3 was activated.Conclusion:This study based on the Zp2 mutation rat model found that:1.ZP deletion may affect protein processing in endoplasmic reticulum,oxidative phosphorylation,TGF-βsignaling pathway and c AMP signaling pathway during oocyte development.2.ZP deletion affected the TGF-βsignaling pathway between oocytes and granulosa cells,led to the down-regulation of the anti-apoptotic gene Bcl2 in oocytes,activated caspase3 and eventually caused oocyte apoptosis.