Epigenetic Modifications And Cellular Fate In Gonadal Differentiation In Monopterus Albus

Both testis and ovary can be produced sequentially in an individual with the same genome during sex reversal in the teleost Monopterus albus,thus determination of cellular fate during ovotestis differentiation will provide new insight into mechanisms of sex reversal.However,how ovotestis is formed remains largely unknown.Here,this thesis reports the first comprehensive single-cell developmental atlas of the model ovotestis.First,single cell RNA sequencing of ovotestis was performed by 10x Genomics.The sequencing depth was over 47,000 reads for each cell from more than 10,000 cells,median detected genes were about 3100 per cell,and total number of detected genes was22184 in ovotestis cells.Over 13 cell clusters were detected by Uniform manifold approximation and projection(UMAP).Based on marker genes,cell types were determined,including germline stem cells,female germline cells,spermatogonia B,spermatocytes,round spermatids,and somatic niche cells(Sertoli and Leydig cells).UMAP analysis provided an overview of cell identities and a roadmap of germline,niche,and stem cell development in ovotestis.Common progenitors of germline stem cells with two states were identified,which revealed their bipotential nature to differentiate into both spermatogonial stem cells and female germline stem cells.Moreover,ovotestis infertility was caused by degradation of female germline cells which was associated with liquid-liquid phase separation of the proteasomes in the nucleus in addition apoptosis and autophagy.Histone-to-protamine replacement in spermatid differentiation was impaired owe to no expression of protomine gene.Notably,signaling pathways in gonadal niche cells and their interaction with germlines synergistically determined distinct cell fate of both male and female germlines.Overall,this thesis reveals a cellular fate map of germline and niche cell development that shapes cell differentiation direction of ovotestis,and provides novel insights into ovotestis development.Epigenetic modification is supposed to be involved in gonadal reversal using the same genome.However,DNA methylation regulation mechanism underlying the gonadal differentiation remains unclear.Liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESI-MS/MS)was used to simultaneously determine endogenous levels of both 5-methyl-2'-deoxycytidine(m~5d C)and 5-hydroxymethyl-2'-deoxycytidine(hm~5d C)during gonadal differentiation.Overall DNA methylation level was upregulated from ovary to testis via ovotestis.As a de novo methylase,dnmt3aa(DNA methyltransferase 3a alpha)expression was also upregulated in the process.Notably,transcription factor Foxa1(forkhead box A1)for dnmt3aa gene expression was determined.Site-specific mutations and chromatin immunoprecipitation showed that Foxa1 can bind to and activate the dnmt3aa promoter.Furthermore,DNA methylation levels of key genes foxl2(forkhead box L2)and cyp19a1a(cytochrome P450,family 19,subfamily A,polypeptide 1a)in regulation of female hormone synthesis were consistently upregulated during gonadal differentiation.These data suggested that dynamic change of DNA methylation modification is associated with gonadal differentiation.