| As an excellent local donkey breed in China,the Dezhou donkey can be divided into two types,the "Sanfen" and the "Wutou",according to the difference in its coat color.The Dezhou donkey is resistant to rough feeding,strong disease resistance and adaptability,and is loved by people everywhere.The excellent genetic traits of breeding donkeys are inherited stably through artificial insemination,and spermatogenesis is responsible for accurate intergenerational paternal genetic information.The testis is an important organ for spermatogenesis in donkeys,and normal testicular development is the basis for the excellent reproductive performance of breeding donkeys.Therefore,it is important to investigate the genetic mechanism and molecular regulatory mechanism of testis development and spermatogenesis in breeding donkeys to select and breed high-yielding and high-quality donkeys.Testis development and spermatogenesis depend on the precise regulation of related genes at the transcriptional and post-transcriptional levels,and non-coding RNAs have been shown to play an extremely important role in transcriptional or post-transcriptional regulation.In this study,Dezhou donkey was selected as the study target,and ncRNAs(lncRNA,miRNA,circRNA)were identified and analyzed at the transcriptome level,with the aim of identifying ncRNAs related to testicular development and spermatogenesis in Dezhou donkey,and trying to analyze the genetic mechanism of donkey testis from the perspective of ncRNAs.At the same time,the testes of Dezhou donkeys at three stages before and after sexual maturation were examined by single-cell transcriptome sequencing technology to construct a single-cell atlas of donkey testis development,identify important genes related to spermatogenesis,and identify a number of donkey testis cell-specific marker genes,which provided an important basis for exploring the transcriptional level regulation mechanism of spermatogenesis and testis development in breeding donkeys.The main results of this study are as follows:(1)By whole transcriptome sequencing of Dezhou donkey testes at three ages(2 months,12 months,and 24 months),46,266 lncRNAs and 63,367 m RNA transcripts were identified,of which 21,845 lncRNAs and 14,109 m RNAs were differentially expressed in juvenile and adult donkey testes,96 lncRNAs and 402 m RNAs were differentially expressed in juvenile and young donkey testes,and 910 lncRNAs and 1,174 m RNAs were differentially expressed in young and adult donkey testes;differentially expressed genes were identified by GO and KEGG enrichment analysis to be enriched in pathways related to spermatogenesis,male reproduction,reproductive processes and testicular development and spermatogenesis;lncRNA-m RNA interaction analysis,an interaction network of 52 DE lncRNAs and 16cis-acting target genes associated with testis development was constructed,in which lncRNA target genes(AMH,ADAM5,DDX18,etc.)were associated with testis development and sexual maturation in donkeys.(2)Sequencing of Dezhou donkey testes at three age stages by whole transcriptome sequencing technology identified 451 known miRNAs,and 135 novel miRNAs.37 miRNAs were differentially expressed in juvenile,young and adult donkeys together,19 miRNAs were differentially expressed in juvenile and young donkeys only,and 25 miRNAs were differentially expressed in 67 miRNAs differentially expressed in young and adult donkeys,and 25 miRNAs differentially expressed in young and adult donkeys only;a total of 315 miRNAs differentially expressed in the three age stages were screened;a large number of target genes were predicted by miRNAs,such as SLC45A4 and TFCP2L1,etc.GO and KEGG enrichment analysis revealed that miRNA target genes were enriched in pathways related to testicular development and spermatogenesis,such as biological reproduction process,fertilization,and sexual reproduction.We also predicted the targeting role of some miRNAs,such as eca-mi R-744,which may affect testis development and spermatogenesis by targeting regulation of SPIN2 B.(3)Sequencing of Dezhou donkey testes at three ages by whole transcriptome sequencing technology identified 12,084 candidate circRNAs.6,409 circRNAs,6,616 circRNAs and 10,031 circRNAs were identified in juvenile,young and adult donkey testis tissues,respectively,with the common 3,908 circRNAs of the three periods.A total of 2,419 circRNAs were differentially expressed in the three periods.8 circRNAs were differentially expressed in juvenile,young and adult donkeys together,29 circRNAs were differentially expressed in juvenile and young donkeys only,1,828 circRNAs were differentially expressed in juvenile and adult donkeys only,and there were 63 circRNAs differentially expressed in young and adult donkeys only.The enrichment analysis by GO and KEGG revealed that they could be significantly enriched in spermatozoa binding to zona pellucida,sperm-egg recognition and other pathways related to male reproduction.A network of 350 circRNAs,29 miRNAs and 39 m RNAs was constructed for interactions,which laid the foundation for the further study of the regulatory mechanism of non-coding RNAs on donkey testis.(4)Transcriptome analysis of donkey testis cells at three ages using scRNA-seq technology identified 26 clusters in Dezhou donkey testis,which contained 4 germ cell types totaling 12 clusters and 6 testis somatic cell types totaling 14 clusters;they were redefined and a total of 9 cell types were defined.A complete single-cell atlas of Dezhou donkey testes was constructed by re-clustering and temporal analysis of germ cell and somatic cell taxa and mapping the differentiation trajectories of each cell subpopulation;a number of potential key genes for spermatogenesis in Dezhou donkeys,including ZPBP,DDX18,THBS3 and AMH,were mined The specific expression and localization of AMH,TNP1,UTF1,ZMYND10 and other genes were verified by immunofluorescence staining,which provided specific marker genes for the identification of various cell types in donkey testes.(5)Transcriptome analysis of normal and sperm-deactivated donkey testes using scRNA-seq technology identified 26 clusters in Dezhou donkey testes,including three germ cell types totaling 18 clusters and five somatic cell types totaling 8 clusters;redefined them to define a total of eight cell types;identified different cell types in donkey testes.The specific marker genes of different cell types in donkey testis were identified,including spermatogonia with high expression of ENTPD3 and NT5 E,and spermatocytes with high expression of NKAPL,CKS2,DBF4,etc.The cell types with significant differences between sperm inactivation and normal sperm groups were identified as macrophages and supporting cells,and significantly different genes C1 QA and RPL10,etc.In conclusion,this study lays the foundation for the study of testicular development and spermatogenesis in Texas donkeys,provides data support for the investigation of sperm inactivation mechanism in Texas donkeys,provides a new scientific basis for further research on molecular breeding in Texas donkeys,and provides a theoretical reference for mammalian spermatogenesis and male sterility related studies. |
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