Histone Arginine Methylase CARM1-mediated H3R26me2 Regulates Development Of Porcine Early Embryos

Pigs are an economically important domestic animal in husbandry and is also a mo del animal in biomedical research.The in vitro production(IVP)of early embryos is a key step of both breeding of elite swines and establishment of biomedical model pigs.However,the developmental competence of IVP porcine embryos is far less than that derived from natural reproduction and IVP embryos from other species.This is main ly due to the inadequate understanding of epigenetic regulatory mechanisms underly ing early embryo development in pigs.Histone arginine methylation as an epigenetic modification plays an important role in cellular differentiation and development in eu karyotes.A recent study showed that H3R17me2 a specifically catalyzed by coactive tor-associated arginine methyltransferase 1(CARM1)was involved in the regulation of preimplantation embryo development in mice.However,the substrate and function of CARM1 in porcine early embryo development remain unclear.The purpose of this study is to elucidate the catalytic substrate and role of CARM1 in porcine early embr yo development.This study will provide theoretical reference for improving the deve lopmental ability of porcine early embryos,the main results are listed as following.The structural analyses of CARM1 gene revealed that it contained 15 exons,located on chromosome 2.The length of m RNA was 3,118 bp,the length of CDS was 1,758 bp,encoding 585 amino acids.The homology analyses of nucleotide and amino acid sequences showed that CARM1 was highly conserved between pigs,humans and mice.Quantitative PCR(q PCR)results indicated that CARM1 m RNA was present in porcin e oocytes and early embryos.The expression levels of CARM1 m RNA were maximu m at the 4-cell and 8-cell embryo stage whereas its expression levels reached a minim um at the blastocyst stage.Immunofluorescence staining analyses indicated that H3R2me2,H3R17me2 a and H3R26me2 modifications exhibited dynamic changes in porci ne oocyte maturation and early embryo development.The effects of CARM1 inhibiti on by a small molecule inhibitor on embryo development were investigated.The resu lts showed that 27 μM CARM1 inhibitor significantly reduced H3R26me2 levels in2-cell,4-cell embryos and blastocysts,but had no effects on the levels of H3R2me2 and H3R17me2a.In addition,CARM1 inactivation resulted in a significant decrease in blastocyst rates.RNAi technique was used to confirm the role of CARM1 in porci ne early embryo development.The results revealed that CARM1 knockdown signify cantly reduced the rate and quality of blastocysts derived from parthenogenetic activa tion(PA)and in vitro fertilization.Additionally,CARM1 knockdown signifycantly de creased H3R26me2 levels in PA 2-cell,4-cell embryos and blastocysts,but did not af fect the levels of H3R2me2 and H3R17me2 a in embryos at the aforementioned devel opmental stages.In conclusion,the expression of CARM1 mRNA and the modification of H3R2me2,H3R17me2 a and H3R26me2 in porcine early embryo development shows dynamic changes.CARM1 specifically catalyzes H3R26me2 in porcine early embryos and participates in regulating early embryo development and blastocyst quality.