Effects Of Vitrification On Morphology, Function And Gene Expression Of Mouse Oocytes

Objective:To study the effect of vitrification on the morphology,function and gene expression of mouse mature oocytes,and to provide experimental data and theoretical basis for the effectiveness and safety of cryopreservation of oocytes.Methods:Mice were injected intraperitoneally with pregnant horse serum gonadotropin(PMSG)and human chorionic gonadotropin(h CG)to stimulate ovulation to obtain mature mouse oocytes,which were divided into fresh group and frozen group.Two kinds of glass were used in the frozen group.Then the Frozen mouse mature oocytes is compared with fresh group,that includes morphology and survival rate of frozen oocytes after resuscitation.After the vitro fertilization of fresh group and frozen resuscitation oocytes,the rate of of fertilization rate and cleavage is also compared.Single-cell transcriptome sequencing was performed on fresh and frozen-recovered oocytes,and differentially expressed genes were compared and analyzed by bioinformatics.Results:1.Observed under a microscope,the oocytes in the vitrified frozen group and the fresh group were round,and there were a few coarse particles in the cytoplasm of some frozen group oocytes.There was no significant morphological difference between the two.The survival rate of the first vitrified frozen oocytes after resuscitation was 78.3%,and the improved survival rate of the second vitrified frozen oocytes after resuscitation was 89.9%.2 The oocytes in the frozen group and the fresh group were observed by scanning electron microscopy,and the complete structure was visible.The oocytes in the fresh group had more protrusions on the surface.Observed by transmission electron microscope,clear nucleus and cytoplasm were seen in both groups,mitochondria,Golgi apparatus and endoplasmic reticulum were seen in cytoplasm.Compared with fresh group,frozen oocytes had looser cytoplasm,more vacuole structure,cortical granules,and less uneven distribution.3.The in vitro fertilization rate of the vitrified frozen oocytes was 38.82%,the cleavage rate was 32.94%,and the blastocyst rate was 18.18%,which (2020)~3indicates that the mature oocytes of mice have the ability to fertilize and develop embryos after cryopreservation.4.The frozen and fresh oocytes were sequenced by single-cell transcriptome,and the sequencing results were obtained for base distribution,gene region distribution,and comparison with known genes in the database,indicating that the data quality is qualified and meets the quality requirements.then the gene expression analysis was performed.The oocytes in the fresh group expressed 18,419 genes,and the frozen group expressed 14,564 genes.There were4,747 genes with significantly different expressions in the two groups,of which 1426 genes were up-regulated and 3321 genes were down-regulated.The genes are annotated and,GO analysis is carried out on 167 genes,which focuses on the cell cycle,cell division,protein ubiquitination,cell stress response and so on.According to KEGG analysis,the differential genes are mainly distributed in related metabolic pathways,RNA transport,and cell cycle.There are many signaling pathways involved as well,including MAPK,Wnt signaling pathway,transcription factor signaling pathway,and Hippo signaling pathway.Conclusions:The improvement of vitrification method can improve the survival rate of mouse oocytes.The oocyte morphology and ultrastructure have no significant changes.The cryopreserved oocytes have the ability to fertilize and develop.There are large differences in the amount of gene expression.This study contributes to the improvement of the freezing efficiency of mouse oocytes,and offers experimental data for evaluating the effectiveness and safety of cryopreservation of mouse oocytes.