Mechanism Of A366 To Improve The Developmental Efficiency Of Mouse Embryos Derived By Round Spermatid Injection

Azoospermia is a important factor leading to male infertility,which occurs in around 1%of the total population of men.In some infertile men,round spermatids are the most mature haploid cells visible during testicular biopsy,and the development of round spermatid injection(ROSI)can help them have their own offspring.Although there are studies about the clinical application of the ROSI technique,there are still problems of low implantation rate,high miscarriage rate,and low live birth rate.In order to improve the development efficiency of ROSI embryos,three small molecule compounds A366,SGC0946 and RG108 were screened from 178 small molecule compounds,which could significantly improve the blastocyst rate of mouse ROSI embryos,and the treated concentration and time of the three small molecules were optimized.The small molecule compound A366,an inhibitor of H3K9me2 methyltransferase G9A,increased the blastocyst rate and live birth rate of ROSI embryos in mice by about 2-fold.At the same time,the weight and litter size of ROSI mice born after incubation with A366 were normal,indicating that A366 is effective and safe,and has great potential for clinical application.To explore the molecular mechanism of A366,we verified the negative effects of redundant H3K9me2 and excessive H3K9me2 methyltransferase G9A on embryonic development by siRNA injection knockdown and mRNA injection overexpression of G9A encoding gene Ehmt2 in mouse ROSI embryos.The incubation of A366 could help round spermatid injection embryos overcome this barrier.In order to analyze the epigenetic state of embryos treated with A366,we also found that the incubation of A366 partially repaired abnormal DNA methylation,chromatin accessibility and transcriptome sites in mouse ROSI embryos by single-cell multi-omics sequencing.It also repaired the expression of minor zygotic genome activation(Minor ZGA)genes.The number of microtuble organizing centers(MTOC)and abnormal phenotype of prokaryotic formation were partially repaired.To further expand the used range of A366,we tried to incubate mouse ROSI embryos with A366 in combination with other small molecule compounds,and found that the combination of A366 and SGC0946 could further improve the blastocyst rate and live birth rate of mouse ROSI embryos.We also studied the B16 cell line after incubating A366.We found that the H3K9me2 modification of B16 cell line was significantly reduced after incubating A3 66,and the expression of some minor zygotic genome activation genes and microtubule associated genes was increased compared with the control group,suggesting that B16 cell line can be used as a complementary model to study the mechanism of A366.In summary,we screened three small molecule compounds A366,SGC0946 and RG108 that can effectively improve the developmental efficiency of mouse ROSI embryos,and analyzed the mechanism of A366,providing a way for clinical optimization of ROSI technology.