Roles And Mechanisms Of Circular RNAs In Porcine Oocyte Maturation And Preimplantation Embryo Development

Circular RNA(circRNA)is a novel class of non-coding RNAs in eukaryotic RNA.In mammals,circRNAs are involved in regulating many biological processes,including spermatogenesis,somatic cell reprogramming,and maintenance of stem cell pluoripotency.In recent years,a large number of circRNAs have been identified in both oocytes and preimplantation embryos of mouse and human.However,the functions and the molecular mechanisms of circRNAs during oocyte maturation and embryonic development remain to be elucidated.In this study,porcine oocytes,cumulus cells,preimplantation embryoswere used as experimental materials to identify the expression of circRNAs by RNA sequencing,and to clarify the expression characteristics and potential roles of circRNAs in oocyte maturation.RNA interference was used to explore the functions and mechanisms of circRNAs in oocyte maturation and early embryonic development.The mainresults were provided as follows.First,oocytes and cumulus cells were collected before and after porcine oocyte maturation.circRNAs in porcine cumulus cells and oocytes were identified by RNA sequencing.Some circRNAs exhibited dynamic changes during oocyte maturation.We found that 7067 and 637 differentially expressed circRNAs in cumulus cells and oocytes during oocyte maturation,respectively.GO functional analysis and KEGG pathway analysis of the differentially expressed circRNAs in cumulus cells and oocytes showed that these circRNAs was mostly related to oocyte maturation,tight junction and c AMP signal pathway.Bioinformatics analysis showed that the differentially expressed circRNAs in cumulus cells and oocytes had a large number of miRNA binding sites.Genome structure analysis showed that maternal circARMC4 expressed in oocytes was formed by spanning back-splicing sites between exon 14 and 15 of ARMC4 gene.RNA interference was used to delete the expression of circARMC4 in porcine oocytes to reveal the regulatory function of circARMC4 in during oocyte maturation.We discovered that circARMC4 knockdown led to a significant reduction in the rate of oocyte maturation and a significant increase in the percentage of misaligned chromosomes,which reduced the developmental efficiency of porcine preimplantation embryos.The analysis of the genomic structure of circRICTOR expressed in cumulus cells showed that circRICTOR was formed by spanning back-splicing sites of exons 9,10,11 and 12 of RICTOR gene.Lentivirus infection-mediated deletion of circRICTOR was used to explore the function of circRICTOR in porcine oocyte maturation.The results revealed that circRICTOR knockdown led to a significant reduction in the area of cumulus expansion and the expression of cumulus expansion related genes,the rate of first polar body extrusion,and developmental efficiency of porcine preimplantation embryos.The expression pattern of circ1073 in porcine early embryonic development was determined by bioinformatics and molecular biology methods.α-Amanitin treatment showed that circ1073 was embryo-derived RNA.RNA interference was used to knockdown the expression of circ1073 to explore the function of circ1073 in porcine early embryonic development.The results showed that circ1073 knockdown could significantly reduce the development efficiency of porcine early embryos and damage the quality of blastocysts.The analysis of gene expression discoved that circ1073 knockdown led to a significant reduction in the expression levels of these genes related with tight junction,adherent junction and cell polarization,anddamaged the barrier function of trophectoderm of blastocysts.Finally,the bioinformatics analysis combined with gene expression analysis showed that circ1073 knockdown led to a significant reduction in the expression levels of miR-128 in porcine preimplantation embryos.In conclusion,circRNA was identified in this study during porcine oocyte maturation and preimplantation embryonic development.It was revealed that maternal circRNA(circARMC4)and cumulus cell-derived circRNA(circRICTOR)regulate the meiosis of porcine oocytes.At the same time,the results demonstrated that circRNA(circ1073)derived from histone demethylase KDM5 B inhibited the expression of miR-128 to regulate the development of porcine preimplantation embryos.These results not only enhance the understanding of molecular mechanisms underlying oocyte maturation and development of preimplantation embryos in pigs,but also provide a theoretical basis for improving the efficiency of porcine oocyte maturation and early embryonic development.