The Function And Regulatory Mechanism Of NAT10 In Mouse Germ Cell And Preimplantation Embryo Development

Human society is facing severe challenges of population aging and declining fertility.At present,the incidence of infertility in China is more than 17%,among which abnormal spermatogenesis,oocyte maturation disorder and preimplantation embryo development arrest are the main causes of infertility.In recent decades,researchers have mainly used mice as a model to carry out a large number of studies on the regulation mechanism of mammalian spermatogenesis,oocyte maturation and preimplantation embryo development.Accumulating evidence suggests that post-transcriptional modifications play important functions in these biological processes.However,little is known about the physiological functions and regulatory mechanisms of mRNA modifications in mammalian germ cells and preimplantation embryo development.N4-acetylcytidine(ac~4C)is the only acetylation modification identified on mRNA recently,which is catalyzed by the writer protein NAT10,and this modification is highly conserved in all species.Studies in somatic cells have shown that mRNA ac~4C modification can enhance mRNA stability and translation efficiency,but whether and how mRNA ac~4C modification is involved in regulating mouse germ cell and preimplantation embryonic development remains unclear.This study explores the physiological function and regulation mechanism of NAT10 in mouse spermatogenesis and"Maternal to zygotic transition"(Maternal to zygotic transition,MZT),which are briefly described as follows:1.NAT10-mediated mRNA ac~4C modification is involved in the regulation of mouse spermatogenesisThis study found that NAT10 was highly expressed in spermatogonia and spermatocytes,and the expression level decreased with the progress of meiosis,and the overall abundance of the ac~4C modification catalyzed by NAT10 gradually decreased during spermatogenesis.To study the function of ac~4C modification in spermatogenesis,this study used Stra8-GFP Cre to construct a mouse model in which Nat10 was specifically deleted in germ cells before entering meiosis,and the male mice were completely infertile.Further analysis found that the loss of Nat10 caused the abnormality of spermatogonia differentiation and meiosis entry;a small number of spermatocytes that could enter meiosis could not complete synapsis and DSBs repair successfully,and there were severe defects in homologous recombination,and spermatogenesis arrested in the pachytene stage.Mechanistic research found that the loss of Nat10 caused a large number of transcripts dysregulated,and the differentially expressed genes were most prominent in the leptotene and zygotene,and the down-regulated genes at this stage were mainly involved in key biological processes such as DSB repair,synapsis,homologous recombination.The overall modification level of ac~4C in the testis decreased after Nat10 deletion,and it was speculated that the decrease of ac~4C caused the down-regulation of the key genes involved in spermatogenesis by affecting the stability of transcripts,and finally result in infertility in male mice.2.NAT10 maintains transcriptome homeostasis during MZTIn this part,we first found that the expression of NAT10 protein gradually decreased during the MZT process.With the activation of the zygotic genome(ZGA),the expression of NAT10 protein gradually accumulates until it reaches the highest level in the blastocyst stage.In addition,the overall modification abundance of ac~4C also gradually decreased during the MZT process.In order to study the function of ac~4C modification in the process of MZT and female fertility,we used Zp3-Cre to construct a mouse model in which Nat10 was specifically knocked out from oocytes of secondary follicles,and the female mice were completely sterile.The phenotypic analysis found that Nat10 deficiency from the primary follicle stage had no significant effect on follicle development,oocyte maturation,ovulation and fertilization,but embryo development was arrested at the 2-Cell stage.These results indicated that NAT10 and its mediated ac~4C modification were involved in the regulation of the MZT process and were required for mouse preimplantation embryonic development.Mechanistic studies have found that the loss of maternal Nat10 resulting in the defect of ZGA and zygotic-dependent mRNA degradation(Zygotic-dependent mRNA decay,Z-decay),and the loss of maternal Nat10 enhances genome instability,resulting in embryo developmental arrest at the 2-Cell stage and ultimately sterility in female mice.