Identification And Expression Regulation Of PiRNA In Mongolian Horse Testis Development And Spermatogenesis

Piwi-interacting RNAs（piRNAs）,as a type of small non-coding RNA,are specifically expressed in male germline cells,mainly distributed in mammalian ovarian oocytes and testicular spermatogonia.piRNA bind to Piwi subfamily protein of Argonaute protein family and regulates the growth and development of germ cells and stem cells,plays a role in transposon silencing,inhibits the formation of heterochromatin,and contributes to maintain DNA integrity,regulates the expression of genes,guarantees the normal development of testis and spermatogenesis.Mongolian horse is one of the best local horse breeds resources in our country,has strong adaptability,strong tolerance,strong resistance,disease resistance and other characteristics.But the number of Mongolian horses in Inner Mongolia area is declining sharply and the varieties are seriously degraded.In this study,piRNA was detected by high-throughput sequencing and bioinformatics,and the distribution and function were analyzed.The piRNA source genes were analyzed by GO function and KEGG Pathway analysis.At the same time,the differential expression analysis of piRNA cluster was carried out to explore the functional mechanism of piRNA in testis development and sperm production of Mongolian horse.The metabolic pathway and piRNAs were significantly different from those of Mongolian horse testis development and spermatogenesis.The effects of piRNA on the reproductive system and reproductive performance of Mongolian horse were further determined..The main results of this study were as follows:（1）Deep sequencing of the two libraries by using Illumia High sequencing technology successfully generated unfiltered raw reads 10,48575 new candidate piRNAs,respectively.There were no significant differences in the number of testicular tissues before and after the upper maturation of the whole small RNA,but the type of small RNA was more abundant in the mature mongolian horse testis.（2）Gene ontology and KEGG pathway analysis of piRNA-generating genes showed that the main GO term these genes enrichment:Intracellular,metabolic processes,cellular protein modification,primary metabolic processes,protein binding,etc.suggesting that these piRNAs are involved in testicular development or spermatogenesis.KEGG enrichment analysis showed that piRNA-generating genes was enriched in various degrees to the Focal adhesion,Phosphatidylinositol signaling system,Adherens junction,etc.In addition,MAPK signaling pathway,Axon guidance,GnRH signaling pathway,Wnt signaling pathway related to the development of the reproductive system;the mucosal spot,the phosphatidylcyclohexanol signal system endocytosis,adhesion and other metabolic pathways directly related to testicular development.In addition,there are MAPK,Axon guidance,GnRH,Wnt and other significant reproductive system related metabolic pathway.（3）The differential expression analysis of piRNA cluster showed that there were 16857 piRNA clusters expressed in all samples before and after sexual maturation,among which the differential expression of piRNA cluster was 7890,of which 3016 were significantly up-regulated and 4874 were significantly down-regulated.There were significant differences in the expression levels of piRNA clusters before and after sexual maturation.It was presumed that these differential piRNA clusters had an effect on the development of testicular tissue before and after sexual maturation.（4）GO function,KEGG Pathway analysis and differential analysis of piRNA cluster showed,some of the piRNA（uniq₂455796,uniq2331522,uniq₂343853,uniq₂3498,uniq₂449807,uniq₂347778 and uniq₂333097）were screened out of differentially expressed genes before and after maturation.And the expression of abundance was detected by real-time fluores-cence quantitative PCR.The expression of piRNAs in the testis tissues was analyzed at different developmental stages.The results showed that seven piRNAs of uniq₂455796,uniq₂331522,uniq₂343853,uniq₂331184,uniq₂449807,uniq₂347778 and uniq₂333097 were expressed in Mongolian horse testis.The expression of uniq₂455796 and uniq₂331522 were significantly different（P<0.05）,and there was no significant difference in the expression of other five piRNAs before and after maturation（P>0.05）.