Functional And Mechanistic Study Of LncRNA 1700008K24Rik In Mouse Spermatogenesis

Spermatogenesis is a crucial process in which male mammals pass down their genes to the next generation,and it is controlled with extreme precision.Long noncoding RNAs(lncRNAs)are highly expressed during spermatogenesis,and their role in regulating this process has gradually become understood.In this study,I conducted functional and mechanistic research on lncRNA 1700008K24Rik,which was found to be highly expressed in the mouse testes.RT-qPCR experiments showed that lncRNA 1700008K24Rik is specifically highly expressed in the mouse testes,and its expression level rapidly increases with the mouse’s sexual development process,maintaining high expression levels after sexual maturity and gradually decreasing after mouse aging.The high expression of 1700008K24Rik in the mouse testes and its expression pattern changing with reproductive development suggest that it may have an important role in mouse spermatogenesis.In this study,by injecting AAV9 viruses carrying specific shRNA into the seminiferous tubules,I successfully knocked down lncRNA1700008K24Rik in the testes.The results showed that when 1700008K24Rik was knocked down in the testes of mice,the testicular morphology exhibited obvious abnormalities,and the number of sperm was significantly reduced,suggesting that lncRNA 1700008K24Rik plays an important role in mouse spermatogenesis.Moreover,the information search from websites such as NCBI blast and piRbase shows that lncRNA 1700008K24Rik overlaps with numerous piRNAs,suggesting that it may be the precursor of these piRNAs.After overexpressing lncRNA and its truncated fragments in the mouse spermatocyte cell line GC-2spd(ts),the levels of related piRNAs were significantly increased,and whether the levels of piRNAs were increased was related to whether the overexpressed fragments contained sequences overlapping with piRNAs.When the overexpressing fragment contained sequences completely overlapping with piRNAs,even incomplete 1700008K24Rik fragments could increase the levels of the related piRNA significantly,supporting the conclusion that 1700008K24Rik is a piRNA precursor.A-MYB,a key transcription factor for piRNA expression in the pachytene stage,was overexpressed in GC-2spd(ts)cells,which not only increased the mRNA expression levels of important proteins in piRNA biogenesis,such as MIWI,TDRD5,and MOV10L1 but also increased the expression levels of 1700008K24Rik and its related piRNAs,suggesting that A-MYB may directly or indirectly participate in the transcription process of 1700008K24Rik.MIWI and MOV10L1 overexpression in GC-2spd(ts)cells can increase the expression levels of 1700008K24Rik-related piRNAs,suggesting that 1700008K24Rik may produce piRNAs through the classical piRNA biogenesis pathway.RNAseq analysis of GC2spd(ts)cells in which 1700008K24Rik was overexpressed showed that genes affected by 1700008K24Rik overexpression not only involve basic cellular processes such as protein synthesis,sugar,lipid metabolism,G protein-coupled receptor,and cAMP signaling pathway regulation but also include some genes that play important roles in spermatogenesis.It is predicted that these reproductiverelated genes may be targeted by 1700008K24Rik-related piRNAs,suggesting that one of the mechanisms by which lncRNA 1700008K24Rik may function is through producing piRNAs to affect the expression of other genes.Additionally,it was found that lncRNA TDRG1,which is homologous to 1700008K24Rik in humans,also produced corresponding piRNAs after overexpression in GC-2spd(ts)cells,indicating that the homology between 1700008K24Rik and TDRG1 is manifested not only in the genomic relative positions and sequences of the gene but also in their ability to produce piRNAs.This study not only revealed the important role of lncRNA 1700008K24Rik in mouse spermatogenesis but also linked it to piRNAs as a potential precursor,broadening the mechanism of action of lncRNAs and deepening our understanding of their function.Additionally,it preliminarily revealed the relationship between the lncRNA TDRG1,the homologous lncRNA of 1700008K24Rik in humans,and piRNAs,indicating that TDRG1 may participate in human spermatogenesis by producing piRNAs.Therefore,it may become a potential drug development target for some male reproduction-related diseases.