Effects Of Vitrification-cryopreservation On Key Gene Expression And Methylation Of Donkey Oocytes During Early Development

The vitrification-cryopreservation of oocytes can not only establish the oocyte bank of fine species and achieve rapid reproduction,but also be an effective means to protect special or endangered species.The aim of this study was to study the differences in gene expression and methylation between vitrified and unvitrified GV stage donkey oocytes cultured at different developmental stages.In this study,GV stage donkey oocytes were matured in vitro for 0 h,20 h and 40 h without vitrification-cryopreservation(control group)and after vitrification-cryopreservation(experimental group),respectively,Single-cell transcriptome and single-cell methylation were sequenced and analyzed in six groups,respectively.The results are as follows:1.Transcriptome sequencing showed that fifteen differentially expressed genes were closely related to their development.Among them,ZAR1 L,DHX29,ELAC1,MED7;SOHLH2 and DAZL are related genes of nuclear transcription and translation,Mitochondrial function related genes POLRMT,CDK5RAP1,TXNIP,SOD2;Gene ERH,MAD2L1,CDK5RAP2-2 related to chromosome assembly of spindle,The genes involved in extracellular matrix action were CPZ and GDF9.Through analysis,the differential expression of these genes is closely related to the low growth rate of frozen oocytes.2.Three genes,WDR49,CSF3 and MPF,The experimental results show that After aftervitrification-cryopreservation,the methylation levels of the three genes were significantly decreased while the transcription levels were increased,which may be related to the development of frozen oocytes.Taken together,the above results show that vitrification-cryopreservation may affect the expression and methylation levels of development-related genes in donkey oocytes,leading to abnormal development or apoptosis of oocytes,This study provides a theoretical basis for improving the maturation rate of oocytes in vitrification-cryopreservation of donkeys and mammal and the development rate of fertilized embryos in vitrified cryopreserved oocytes of donkeys and mammals.