| The basis of fish reproduction is the formation of gametes,also known as gametogenesis,including spermatogenesis and oogenesis.Gametogenesis is a highly coordinated process,which depends on the regulation of autocrine/paracrine hormones and growth factors.However,the molecular mechanism regulating fish gametogenesis is still lack of in-depth research,especially in fish with natural sex reversal.In this study,we obtained high-resolution single-cell transcriptome profiles of gonadal cells in male,pseudomale and female of Chinese tongue sole,in order to reveal the dynamic gene expression regulation during gametogenesis and the spermatogenesis mechanism of pseudomale as well as the conserved transcriptional features of gametogenesis across vertebrates.The main research contents and results are as follows:1.Analysis of spermatogenesis of male and pseudomale of Chinese tongue sole based on single-cell sequencing.According to the expression pattern of known marker genes,five germ cell types(including undifferentiated spermatogonia,differentiated spermatogonia,preleptotene spermatocytes,pachytene spermatocytes and sperm)and six somatic cell types(including Sertoli cells,Leydig cells,T cells,NKT cells,B cells and Erythrocytes)were identified from the testis of Chinese tongue sole.In addition,we found that the key genes of spermatogenesis,such as gfra1,spo11,sycp1,sycp3,dnajb13,tekt1 and gata4,have similar expression patterns in human,mouse and Chinese tongue sole testis cells.Additionally,we reconstructed the differentiation process of male and pseudomale germ cells based on a pseudotime analysis,and revealed the dynamic gene expression characteristics during spermatogenesis.Furthermore,we found that male and pseudomale testis had the same cell types and showed similar gene expression pattern by the comprehensive comparative analysis of single-cell transcriptome data of male and pseudomale testis.However,compared with male fish,the proportion of undifferentiated spermatogonia in pseudomale fish is higher,while the proportion of differentiated spermatogonia and preleptotene spermatocytes is lower.The expression level of G1/S phase genes in pseudomale undifferentiated spermatogonia and preleptotene spermatocytes was lower than that in male fish,but there is no significant difference in the expression of G2/M phase genes.The expression of genes involved in cell cycle activation(including cdc45 and meioc)was significantly down-regulated in all germ cells of pseudomale except sperm,while autophagy related gene ykt6 and apoptosis promoting gene znrf3 were significantly up-regulated in differentiated spermatogonia and preleptotene spermatocytes of pseudomales.By analyzing the gene expression of male and pseudomale undifferentiated spermatogonia,we found that "cell proliferation","MAPK family signaling cascades" and Ca SR were up-regulated in pseudomale undifferentiated spermatogonia,while "calcium signaling pathway" was down regulated.By comparing and analyzing the spermatogenic microenvironment of male and pseudomale,we found that the proportion of Leydig cells in pseudomale was significantly lower than that of male,but there was no significant difference in the proportion of Sertoli cells.Through gene expression analysis,we found that the signal pathways and genes related to androgen synthesis(including hsd3b1,cyp17a1,cyp11a1,hsd11b2 and hsd17b1)were significantly down regulated in pseudomale Leydig cells,and the signal pathways and genes related to Sertoli cell function were significantly down regulated in pseudomale Sertoli cells.In addition,we also found that the expression level of Z chromosome specific genes in testis cells of male was higher than that of pseudomale,but there was no significant difference in autosomal genes.2.Analysis of oogenesis of Chinese tongue sole based on single-cell sequencing.We identified three germ cell types(including Early oocytes,Mitotic oocytes and Oocytes)and five somatic cell types(including Follicle cells,Endothelial cells,Macrophages,T cells and Erythrocytes)and revealed the gene expression characteristics of each cell type.We used pseudomale analysis to reconstruct the oogenesis and found that Early oocytes,Mitotic oocytes and Oocytes followed a continuous development trajectory.Notably,many oogonia markers,mitotic related genes and meiosis related genes were enriched at different stages of the pseudotime trajectory.Furthermore,we identified four granulosa cell subtypes(GC1,GC2,GC3 and GC4)and one theca cell type(TC),and revealed the gene expression characteristics of each cell subtype.In addition,we performed an unbiased cell communication to investigate the complex interactions involved in fish oogenesis.We found that bidirectional communication occurred between germ cells and granulosa cells,while germ cells and theca cells only showed unidirectional regulation from theca cells to germ cells.The ligands and receptors of TGF-β signaling pathway and Notch signaling pathway were expressed in distinct ovarian cells,and the expression pattern was consistent with that in mammals.In addition,we found ovulation related genes lhcgr,pgr,ptger4 b,adamts1 and ecm1 were specifically expressed in different granulosa cell subtypes,indicating the synergistic regulation of different granulosa cell subtypes in the ovulation of fish.Finally,we compared our data with the reported sc RNA-seq datasets in macaques and found that most of the differentially expressed genes in the ovarian germ cells and granulosa cells of Chinese tongue sole were homologous to macaques,and these homologous genes were significantly enriched in the biological processes related to oogenesis.In addition,several homologous genes related to oogenesis,such as zar1,sall4,ybx2,dnd1,meiob,gdf9,foxl2,fshr,wt1,hsd17b1,cdh1 and bmpr2 showed similar gene expression patterns in ovarian cells of Chinese tongue sole and macaques.This study provides a comprehensive high-resolution transcription map of fish gonadal cells based on single-cell sequencing.We analyzed the heterogeneity of Chinese tongue sole gonadal cells and revealed the dynamic changes of gene expression patterns during fish gametogenesis under single-cell resolution,providing a reference for the in-depth study of fish gametogenesis.Through the comparative analysis of spermatogenesis between male and pseudo male fish,the differential phenotypic characteristics and gene expression characteristics in the process of spermatogenesis between male and pseudo male fish were analyzed.At the same time,we identified four granulosa cell subtypes and one theca cell regulating oogenesis and revealed the interaction between these cell subtypes and germ cells as well as their synergistic regulation in the process of ovulation.We investigated the conserved transcriptional characteristics of oogenesis in vertebrate evolution combined with the single cell transcriptome data of macaque ovaries.These findings can provide a theoretical basis for the artificial breeding of Chinese tongue sole and the cultivation of all female seedlings.At the same time,they are of great significance to reveal the fertility mechanism of sex reversal population widely existing in nature.In addition,it is helpful to understand the adaptability of reproductive strategies of lower vertebrates and the common pattern of gametogenesis of vertebrates,and provide a theoretical reference for the related research of fish reproductive biology. |
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