| ObjectiveBisphenol A(BPA),a typical environmental endocrine disruptor which is ubiquitous within the environment.Based on its high temperature resistance,high strength,good toughness and other characteristics,it is mainly used in the production of food and beverage containers,plastic water cups,paper cups and plastic packaging bags.People are exposed to BPA mainly through skin,inhalation,and digestive system.BPA has been shown to cause the damage to normal testicular development and spermatogenesis,however,the potential mechanism has not been elucidated.Single-cell RNA sequencing(scRNA-seq)is a novel technique for high-throughput sequencing analysis of the transcriptome and epigenome at the single-cell level,which reveals the gene expression of individual cells and reflects the intercellular heterogeneity.BPA was used as a model of male reproductive damage to investigate the adverse effect of BPA on testis development and the molecular mechanism of abnormal spermatogenesis in mice,combined with single-cell sequencing technology.This study will provide a new target for the prevention and treatment of abnormal spermatogenesis and male infertility caused by BPA.MethodsThe male pubertal mice were randomly divided into five groups: the blank control group,the solvent control group,5mg/kg BPA,50mg/kg BPA,and100mg/kg BPA,and each group of mice was given gavage one day apart for12 weeks(from P28 to P112)(Postnatal 28-day old,P28),respectively,to construct a BPA pubertal exposure model.The semen in the epididymal tail and testicular tissue were taken and the sperm quality parameters(total sperm counts,total sperm density,motile sperm count,and immotile sperm counts)were detected by semen quality analyzer in each group.Testicular histomorphological changes were observed by hematoxylin and eosin staining(HE).Single-cell sequencing analysis was performed on representative testicular tissue from the blank control group and the 100mg/kg BPA group to further explore the potential molecular mechanism of testicular injury and abnormal spermatogenesis induced by BPA exposure.In this study,single-cell sequencing data were first subjected to quality control and filtered out unqualified cells.After the dimensionality reduction clustering of these cells,each cell population was annotated according to the differential expressions of the marker genes.Next,the differentially expressed genes(DEGs)between two groups were screened for Gene Ontology(GO)enrichment and(Kyoto Encyclopedia of Genes and Genomes)KEGG signaling pathway analysis.Then,germ cell clusters were extracted separately for further subpopulation analysis to clarify the molecular mechanism of reproductive toxicity.The round and elongating spermatids were divided into different developmental stages after the pseudo-time analysis,and the subpopulation analysis was performed for the germ cell populations.In addition,SCENIC analysis was used to explore transcription factors in various germ cells that regulate testicular injury.Finally,cell-cell communication analysis was used to find signaling pathways between various cell populations that were associated with abnormal spermatogenesis.Results(1)Effects on semen quality of mice: Compared with the blank control group,sperm motility trajectories,total sperm count,sperm concentration,and motile sperm count in 50mg/kg and 100mg/kg BPA exposure groups were significantly decreased(P<0.05),while non-motile sperm count was significantly increased(P<0.05).(2)Effects on testis development and morphology: Testis organ coefficient of 100mg/kg BPA exposure group was significantly lower than that of the blank control group(P<0.05).In the blank control group and the solvent control group,HE staining results showed no significant difference in the morphology of the seminiferous tubules.Compared with the blank control group,50mg/kg and100mg/kg BPA exposed groups showed an increased trend in the diameter and lumen diameter of testicular seminiferous tubules,but there was no statistical significance(P>0.05).The thickness of spermatogenic tubule epithelium was significantly decreased in the 100mg/kg BPA exposure group(P<0.05).Based on the above evidence,in this study,representative testicular tissues of blank control group and 100mg/kg BPA exposure group were selected for scRNA-seq sequencing and subsequent data analysis.(3)Cell identification and enrichment analysis were performed on testicular tissue: Based on the differential expression of marker genes,testicular tissue was defined as 10 cell types,including 4 germ cells(spermatogonia,spermatocyte,round and elongating spermatids)and 6 somatic cells(Sertoli cells,fibrocytes,Leydig cells,endothelial cells,innated lymphocytes,and macrophages).Compared with the blank control group,the number of germ cells in the BPA exposure group was decreased.The number of Leydig cells,fibroblasts,macrophages,and innated lymphocytes was increased in the BPA group,while the number of endothelial cells and Sertoli cells increased.(4)Effects on spermatogonia: The spermatogonia was divided into undifferentiated spermatogonia and differentiating spermatogonia by subpopulation analysis.Compared with the control group,the proportion of differentiating spermatogonia decreased in the BPA exposure group.Enrichment analysis of common DEGs in two spermatogonia showed that were both enriched in biological processes such as spermatogenesis,male gamete generation,and germ cell development.DEGs of undifferentiated spermatogonial cells were enriched in regulation of m RNA metabolic process,protein and purine metabolism.DEGs of differentiating spermatogonia were enriched in m RNA metabolism,chemical carcinogenesis and oxidative phosphorylation.(5)Effect on spermatocyte: spermatocyte was subgroup analyzed and divided into four developmental stages stage,such as leptonema,zygonema,pachynema,diplonema.Compared with the control group,the proportion of diplonema spermatocyte decreased in the BPA exposure group.A common differential gene Ide was found in the four spermatocytes,which was also a common differential gene in both undifferentiated and differentiating spermatogonia.Ide participates in regulating the differentiation and proliferation of germ cells enrichment analysis of DEGs showed that four spermatocyte subsets were mainly enriched in biological processes such as protein regulation and metabolism and spermatogenesis.(6)Effects on round and elongating spermatids: Based on pseudo-time series analysis,the two types of sperm cells were divided into early spermatids and advanced spermatids.DEGs of round and elongating spermatids were mainly enriched in protein metabolism,oxidative phosphorylation,chemical carcinogenesis,spermatogenesis and other biological processes.(7)Effects on gene regulatory network of germ cells: After BPA exposure,24 regulators with significant changes in AUC activity were screened from spermatogonia,39 regulators form spermatocytes,19 regulators from round spermatids and 5 regulators from elongating spermatids.Combined with differential gene analysis,seven differentially expressed transcription factors(EGR1,THAP1,KDM5 B,CREB3L4,SOX30,POLE3,YBX1)were found to be involved in regulating the process of germ-cell damage.(8)Effects on cell population interactions: BPA exposure enhances cell interactions.Among them,Sertoli cells,fibrocytes,and macrophages affect spermatogenesis of germ cells through two ligand-receptor pair,CADM1-CADM1 and APP-CD74.ConclusionsIn summary,we discussed that(1)BPA exposure interfered with normal testicular development and spermatogenesis,causing decreased sperm quality and changes in tissue morphology;(2)Decreased the number of germinal cells,inhibited spermatogonal differentiation,meiosis and transformation into mature cells,and changed the activity of transcription factors(EGR1,THAP1,KDM5 B,CREB3L4,SOX30,POLE3,YBX1),which were involved in the regulation of abnormal spermatogenesis;(3)Decreased the number of sertoli cells,increased the number of macrophages and fibrocytes,and affected spermatogenesis through CADM1-CADM1 and APP-CD74 ligand receptor pair.This study will contribute the understanding in the mechanism of male reproductive toxicity of BPA and the preventing the effects of BPA on development of male gametes. |
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