Hypoxia Affects Mice Oocyte In Vitro Maturation And Regulates Transcriptome And MiRNA Expression Profiles Of Cumulus Cells

The lack of blood vessel distribution inside the mammalian follicles causes the oocytes and cumulus cells to be in a hypoxic state(for humans,the oxygen tension is about 1.3%-5%).Studies have shown that hypoxia can affect the level of histone methylation in cells.Histone methylation,as an important epigenetic modification,plays an important role in regulating gene expression.Cumulus cells and oocytes are known to forms are functional syncytia complex called cumulus oocyte complex(COC).The exchange of materials and information between them is essential for the development of follicles and oocytes.Changes in gene expression of cumulus cells affect the state and function of the oocyte.The quality of oocytes will be affected by gene expression profile alterations of cumulus cells.However,there is no report about the regulation of hypoxia on histone methylation of cumulus cells.Other studies have shown that hypoxia can regulate expression profile of miRNA in cells.miRN A is a non-coding RN A that can regulate protein synthesis after transcription of its target gene,thereby playing an important role in cell growth,differentiation,apoptosis,and substance synthesis.In this study,immunofluorescence,Western blot and other methods were used to detect changes in partial phenotypes of mouse oocytes cultured in vitro under hypoxic partial pressure and changes in H3K4me3,H3K27me3,and HIF1α levels of cumulus cells under different oxygen tension.At the same time,transcriptomic analysis was performed on cumulus cells to explore the changes in transcriptome and miRNA expression profiles under hypoxic conditions.After the above research,the paper initially revealed the effects of hypoxic oocyte maturation and cumulus cell gene expression.The research results provide theoretical guidance for the improvement and optimization of the oocyte culture system in vitro.(1)Effects of different oxygen tension on the vitro maturation of oocytes and histone methylation of cumulus cellsIn this study,the maturation rate of oocytes under different oxygen tension(20%,5%,1%)was counted.The results showed that as the oxygen concentration decreased,the maturation rate of oocytes also decreased significantly(P<0.05);In order to explore the possible reasons for the decrease in the maturity rate,we performed immunofluorescence staining of oocytes at 5%and 20%oxygen concentration,and found that different oxygen tension did not affect the expression and distribution of oocyte Tubulin and Actin,ROS level increased slightly(P>0.05);while hypoxia would increase the expression of HIF 1α in oocytes.Next,the paper studied the effect of hypoxia on HIF1α protein expression and histone methylation modification in cumulus granulosa cells,and found that compared with normoxic conditions,the expression of HIF1α in cumulus cells increased under hypoxia,and the levels of H3K4me3 and H3K27me3 increased.(P<0.05).These results indicate that hypoxia will affect the development of oocytes,and will also affect the histone modification level of cumulus cumulus cells,and hypoxia will increase the level of HIF1α in oocytes and cumulus cells.(2)RNA-Seq analysis of the effect of different oxygen tension on gene expression of cumulus cellsIn this experiment,a total of six transcriptome sequencing libraries were constructed,with(20%,5%)each having three replicates,and an average of about 38M(millions)of raw reads were obtained.Cutadapt was used to filter out unqualified sequences for the next step.For analysis,Hisat software was used to compare the sequencing data to the reference genome,and String Tie software was used to assemble transcripts and predict expression levels.A total of 141767 transcripts were obtained,and 55450 genes were obtained.Differential expression analysis obtained a total of 1343 differentially expressed genes,of which 1018 were up-regulated and 325 genes were down-regulated.The hierarchical clustering analysis of the differentially expressed genes found that the three samples under the same processing conditions can be well grouped together.This shows that the sample has good repeatability and high reliability.By performing GO functional annotation and KEGG pathway enrichment analysis on the differentially expressed genes,626 GO entries were screened for significant enrichment,mainly including the glycolysis process,hypoxic response,and regulation of transmembrane transport.There are 36 KEGG pathways that are significantly enriched,and the main enrichment-related pathways are:glycolysis and gluconeogenesis,HIF signaling pathway.Ten differentially expressed mRNAs were randomly selected for qRT-PCR verification,and their expression trends were consistent with sequencing results.(3)miRNA sequencing analysis to study the effect of hypoxia on cumulus cellsIn this study,a total of 6 sequencing libraries were constructed,and an average of 11.5M(millions)of raw data was obtained.After data filtering,an average of 6.75M(millions)of effective data was obtained,and the data volume met the requirements for further analysis.Length analysis of the obtained miRNA,the length is mostly between 20-24nt.A total of 14 differentially expressed miRNAs were screened,of which 6 were up-regulated and 8 were down-regulated.The cluster analysis of the obtained miRNAs,the three replicates of the two groups of experiments can be well grouped together.Enrichment analysis of miRNA target genes,the enriched GO items are mainly protein binding(Protein binding),metal ion binding(Metal ion binding)and G protein-coupled receptor activity(G protein-coupled receptor activity).The involved KEGG pathways are mainly olfactory transduction,calcium signaling pathway,phosphatidylinositol signaling system,Wnt signaling pathway and so on.Four miRNAs were selected for verification by qRT-PCR,and the expression trend was consistent with the sequencing results.In summary,we explored the effect of hypoxia on the in vitro maturation of COCs in mice and the possible mechanism,that is,during the in vitro maturation of COCs,hypoxia partial pressure will hinder their maturation.At the same time,hypoxia will change the degree of histone methylation of CCs and the level of HIF1α.Transcriptome sequencing technology proved that hypoxia will significantly affect the gene expression of CCs.This experiment provides basic data for studying the effects of hypoxia on mammalian reproduction.