| Introduction:The oviducts and efferent tubules are closely related to infertility.It is known that impaired oviduct patency accounts for 35%of cases of low fertility and infertility.Recent mouse studies have shown that reduced efferent tubular function is associated with obstructive oligospermia and fertility issues.The oviduct is a tubular structure that connects the uterus and ovaries in female mammals and is the only channel for transporting eggs.According to its anatomical structure,it can be divided into the isthmus,ampulla,and infundibulum,which respectively play roles in sperm transportation and storage,gamete binding,and egg pickup.The efferent tubule is the tubular structure connecting the testis and epididymis in male mammals.Its main function is to transport sperm and concentrate the seminiferous tubule fluid.Most current studies of the efferent tubule are limited,such as looking at the function and localization of a protein or the localization of a specific cell type.Therefore,it is very important to study the biology of the efferent tubule at the level of individual cells.The efferent tubules are one of the rare structures with motile cilia and share epithelial cell types with the fallopian tubes.During each menstrual cycle,follicular fluid that is high in reactive oxygen species and inflammatory agents damages the oviduct epithelium.The epithelium of the oviduct must be similar to the tracheal epithelium in order to function properly,and stem cells are also present to repair damaged epithelium.The types of stem cells,marker genes,and differentiation trajectories in the oviduct epithelium are yet unknown.It is unknown whether the differentiation trajectories of the epithelial cells at various anatomical sites of the oviduct follow the same pattern,given their substantial variability.Correspondingly,are stem cells also present for the regeneration of tissue when the efferent tubule epithelium is damaged?It is known that infertility in couples of childbearing age is caused by oviduct factors or efferent tubule factors.The failure of the oviduct and efferent tubule epithelium to operate appropriately is one of the key causes.Therefore,it is very important to understand the homeostasis of the epithelial cells of the oviduct and the efferent tubule.Only by comprehending the process of epithelial cell healing in physiological settings can we understand and detect aberrant pathological states.Recently,single-cell sequencing technology has been widely used in the identification and functional study of various tissue cell types,and it has unique advantages in developmental biology and stem cell differentiation.Therefore,this study intends to use single nuclear sequencing technology to carry out the following studies:1)analyze the cell composition of the oviduct and the efferent tubules and draw the single cell maps of the two tubules,respectively;2)analyze the function,heterogeneity,differentiation locus,genes,and transcription factors involved in the regulation of the efferent tubule and tubal epithelial cell subsets,identify the types of intraepithelial dry cells,and reveal the mechanism of epithelial homeostasis in the two tissues;3)The cellular and intercellular communication networks in the efferent tubules and oviducts were analyzed to preliminarily reveal the regulatory role of non-epithelial cells in the process of epithelial cell differentiation.Materials and methods:1.Experimental materials:efferent tubule tissue of male mice and oviduct tissue of female mice.2.Experimental methods:next-generation sequencing,sequencing data aligment and quantification,elimination of environmental pollution RNA,sequencing data quality control and standardization,PCA dimensionality reduction,tSNE and UMAP clustering results visualization,difference analysis,cell type annotation,GO and KEGG,pseudotime analysis,transcription factor regulation analysis and intercellular communication network analysis.Results:1.Quality control results of single nuclear sequencing and data analysis The output results of the Cell Ranger software showed that the quality control indexes of single nuclear sequencing(number of target sequences,median gene,effective barcodes,UMIs,Q30,etc.)were qualified.2.Analysis of oviduct sequencing data2.1 Single cell map of the oviductNine cell types were found in the isthmus of the oviduct,seven in the ampulla,and five in the infundibulum.The proportion of ciliated cells in the infundibulum part was the highest,the ampulla was the second,and the isthmus was the least.The oviduct secretory cells showed the opposite trend,and their heterogeneity increased gradually from the infundibulum part to the isthmus.GO enrichment analysis showed that many genes highly expressed in non-epithelial cells were enriched in biological activities related to extracellular matrix,cell proliferation,differentiation,and migration.2.2 Oviduct epithelial cell heterogeneityStem cells expressing Lgr4 and Sox5 and mature secretory cells expressing Pax8 and negative for Lgr4 were found in the oviduct epithelium.At the same time,transitional cells(ciliated precursor cells)expressing secreting cell marker genes(Ovgp1,Wfdc2,Clu)and ciliated cell marker genes(Foxj1,Dnah12,Cfap43)and ciliated cells expressing highly Foxj1,Dnah12,Cfap43,etc.Stem cells were found in all three parts of the oviduct,mature secretory cells were found mainly in the isthmus,and ciliated cells were found mainly in the ampulla and infundibulum.2.3 Development and differentiation of oviduct epithelial cellsBy pseudo-time analysis,it was found that epithelial stem cells in the oviduct have two fates of differentiation:secretory cells and ciliated cells.We found that the isthmus stem cells mainly differentiated towards secretory cells,and the ampulla and infundibulum stem cells mainly differentiated towards ciliated cells.The PAGA trajectory plot indicates that secretory cells also have a potential differentiation relationship with ciliated cells.In the process of stem cell differentiation into ciliated cells,hypervariable genes related to the development of the urogenital system,regulation of membrane potential,extracellular matrix,Wnt signaling pathway,regulation of epithelial cell proliferation,pattern specialization process,and ciliary assembly were found.In the process of differentiation of stem cells into secretory cells,hypervariable genes related to positive regulation of nervous system development,cell junction assembly,positive regulation of protein transport,and calcium homeostasis were identified.2.4 Analysis of transcription factors in oviduct epithelial cellsWe used transcription factor analysis to find the top five transcription factors with specific differential scores for oviduct epithelial cell subsets and calculated their relative activities.These transcription factors may play a key role in the functional realization,development,and differentiation of epithelial cells.2.5 Communication network analysis of oviduct cellsFibroblasts in the oviduct are dominant in regulating other cell types.A total of 10 interacting signaling pathways were found between oviduct cells,the first three being FGF,IGF,and BMP,which are mainly emitted by fibroblasts and mostly act on epithelial cells.The most significant ligand-receptor pair in this communication network is Fgf1-Fgfr2.These results indicate that fibroblasts play an important role in the regulation of epithelial cells.3.Analysis of efferent tubule sequencing data3.1 Efferent tubule single cell mapFive cell types were identified in the efferent tubules,and secretory cells accounted for the highest proportion.We found some new marker genes in efferent tubule-ciliated cells and secretory cells.Subsequent annotation of different cell types revealed that,consistent with the oviduct,the hypervariable genes in non-epithelial cells were also enriched in biological processes related to proliferation,differentiation,and migration.3.2 Heterogeneity of efferent tubule epithelial cellsIn the efferent tubule,we found stem cells expressing stemness genes such as Sox6,Kit,and Itga6,secretory cells,which in a stress state express Fos,Jun,and Pax8,and secretory cells expressing Esr1 and Zbtb16,transitional cells expressing Wfdc2,Clu,Dynll1,and Tubb4b,and ciliated cells expressing Foxj1,Cfap43,and Dnah12.3.3 Development and differentiation of efferent tubule epithelial cellsThrough pseudo-time analysis,it was found that the stem cells in the efferent tubule epithelium could differentiate into ciliated cells through transitional cells.According to the results of the quasi-temporal gene expression,we found that the highly variable genes related to sex differentiation,arterial development,membrane potential regulation,and ciliary assembly may be involved in the process of differentiation.3.4 Transcription factor analysis of efferent tubular epithelial cellsWe used transcription factor analysis to find the top five transcription factors with specific differential scores for a subset of efferent tubular epithelial cells and calculated their relative activities.These transcription factors may play a key role in the functional realization,development,and differentiation of epithelial cells.3.5 Communication network analysis of efferent tubular cellsIn the efferent tubules,we found that fibroblasts were equally dominant in regulating other cell types.A total of 11 interacting signaling pathways were found in efferent tubule cells,most of which were transmitted from fibroblasts and mainly received by endothelial cells and epithelial cells.Igf1-Igfr1 is the most significant ligand-receptor pair in this communication network.Efferent tubular epithelial cells are regulated by five external signals,including IGF,NRG,GAS,ncWNT,and BMP.These results indicate that fibroblasts play an important role in the regulation of epithelial cells.4 Differential gene expression analysis between efferent tubule and oviduct ciliated cells Compared with oviduct ciliated cells,170 genes were up-regulated in efferent tubule ciliated cells,which were related to cognition,cell junction assembly,amide transport,calcium-dependent cell-cell adhesion through plasma membrane adhesion molecules,and regulation of the Wnt signaling pathway,and 138 genes were down-regulated,which were related to regulation of centriole migration and maintenance of intracellular position.Conclusions:1.Various subpopulations of oviduct epithelial cells exist,including stem cells,secretory cells,ciliated precursor cells,and ciliated cells.They are distributed differently in different parts of the oviduct.2.Oviductal epithelial stem cells have two fates of differentiation:secretory cells and ciliated cells.The stem cells in the isthmus of the oviduct differentiate mainly in the direction of secretory cells,while those in the ampulla and fimbria differentiate mainly in the direction of ciliated cells.3.Five cell types were identified in the efferent tubule,including secretory cells,ciliated cells,fibroblasts,endothelial cells,and macrophages.4.There are sternness secretory cells in the efferent tubule epithelium that have the destiny to differentiate into ciliated cells.5.Multiple hypervariable genes and transcription factors are involved in the differentiation of stem cells in the epithelium of the oviduct and efferent tubule,respectively.6.Fibroblasts play a major role in regulating epithelial cells in the oviduct and efferent tubules.7.There are differentially expressed genes related to cognition,cell connection and assembly in ciliated cells of efferent tubules and oviduct. |
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