The Epigenetic Landscape Of Development Of Mouse Embryos Derived By Round Spermatid Injection

Patients with severe azoospermia have no mature sperm or elongating spermatid in the testis and epididymis and therefore cannot obtain genetic offspring by intracytoplasmic sperm injection(ICSI).Nevertheless,Round spermatid injection(ROSI)is an important treatment to help some of azoospermia patients who still have spermatid to obtain genetic offspring through assisted activation of oocyte after injection of round spermatid.Some progress has been made in obtaining healthy offspring in mice,rabbits,monkeys,and humans using ROSI techniques,however,there are still problems of low implantation rate,high miscarriage rate,and low live birth rate.The molecular mechanisms leading to the low developmental potential of ROSI embryos are still unclear,thus to find the key obstacles in the development of ROSI embryos is particularly critical and important for improving the developmental potential of ROSI embryos and promoting the clinical application of this technology in the field of assisted reproduction.In order to clarify the development of mouse ROSI embryos throughout their development,we established a stable system of ROSI,embryo culture and transplantation.Compared with ICSI embryos,ROSI embryos have low compaction rate,low blastocyst rate,low implantation rate and low live birth rate.What’s more,at pronuclei stage,a higher proportion of ROSI embryos appeared to be with single pronuclei,which could not develop to blastocyst.This suggests that the low developmental rate of ROSI embryos may be related to abnormal pronuclei formation.To understand the molecular mechanisms of RO SI embryo development,for the first time,we describe the dynamic change of gene expression,chromatin accessibility,and DNA methylation during preimplantation development of mouse ROSI embryos and ICSI embryos by single-cell multi-omics sequencing.Our analysis shows that ROSI embryos have abnormal downregulation of Minor zygotic genome activation(Minor ZGA)genes associated with cytoskeletal organization in ROSI PN3 embryos.The diameter of male pronucleus of ROSI PN3 embryos are confirmed to be small by white light and immunofluorescence observation.At the same time,the number of γTUBULN dots in ROSI embryos decrease significantly,which may affect microtubules and other cytoskeletal structures,and thus affect the structural remodeling of male pronucleus and further affecting the developmental potential of ROSI embryos.Meanwhile,abnormal DNA methylation and chromatin accessibility mainly exist in pronucleus stage(PN3).Part of the abnormal expression of Minor ZGA genes associated with hyper DNA methylation and low chromatin accessibility,which also enrich in the biological event of actin cytoskeleton organization.These results suggest that there is a close relationship between the chromatin remodeling abnormalities,down-regulated minor ZGA genes and cytoskeleton organization in ROSI pronucleus embryos.In summary,we describe the single-cell gene expression,DNA methylation and chromatin accessibility maps of mouse ROSI embryos and ICSI embryos for the first time,and compare the differences in DNA methylation,chromatin accessibility and transcription between them.We find that the abnormal activation of minor ZGA and the abnormal epigenetic remodeling in ROSI pronucleus embryos may be an important cause of the poor developmental potential of ROSI embryos,which of vital importance for the further study of the epigenetic mechanism of ROSI embryo development.