The Function Study Of The Testis-specific LncRNA In Spermatogenesis

Background: Long non coding RNA(lncRNA)are a class of regulatory RNAs with a length of more than 200 bp.Lnc RNA play important roles in the regulation of various life activities and the occurrence and development of diseases.However,it is not clear whether lncRNA are involved in mammalian spermatogenesis.Spermatogenesis is a fine regulated biological process that eventually produces mature sperm,but its mechanism is largely not elucidated.In this study,two testicular specific lncRNA lnc240(NONCODE ID: NONMMUT049240.2),Gm31453 and its potential coding gene Cpa5(carboxypeptidase A5,Cpa5)were selected to investigate the role and mechanism of lncRNA in spermatogenesis under the normal physiology and sterility model induced by drug.This study will provide a new perspective for understanding the regulation mechanism of spermatogenesis.Method: Spermatogenic cells at different stages were sorted by flow cytometry to investigate the expression levels of selected genes.lnc240,Gm31453 and Cpa5 KO mice were constructed with CRISPR /Cas9 technology to study the spermatogenesis and fertility of KO mice.Hematoxylin-eosin staining(HE)were applied to analyze the development of testis and epididymis.Sperm morphology and motility were measured by computer aided sperm analysis system.The role of lncRNA in spermatogenesis under triptolide stress was addressed by high-throughput sequencing and bioinformatics analysis.The gene expression levels were evaluated by q PCR in KO mice.Result: lnc240,Gm31453 and coding gene Cpa5 that may be targeted regulated by Gm31453 in Cis manner,were specifically expressed in secondary spermatocytes,round and elongated spermatids.lnc240,Gm31453 and Cpa5 KO mice showed no significant differences in testis and epididymis development,sperm concentration and motility,and fertility compared with wild-type mice.Since Gm31453 is an interacting molecule of STRBP and may regulate the expression of Cpa5,we analyzed the effects of Gm31453 on the expression of STRBP and Cpa5.The results showed that deletion of Gm31453 resulted in down-regulation of CPA5 and up-regulation of STRBP.In view of the absence of lncRNA did not show obvious reproductive phenotype under normal physiological conditions,we continued to investigate the effect of lncRNA on spermatogenesis in an oligo-atheno-terato-spermia mouse model caused by adverse stress.After 35 days of intragastric administration of triptolide in mice,testis development was significantly abnormal,teratospermia appeared in epididymis and sperm count was dramatically reduced.High-throughput sequencing showed that many lncRNA were dysregulated by triptolide.The GO and KEGG function enrichment analysis of differentially expressed m RNAs and lncRNAs target genes revealed that a variety of spermatogenesis related biological processes and signaling pathways were markedly affected.However,triptolide at the same dose displayed less reproductive toxicity in Gm31453 KO mice compare WT.Conclusion: Single KO of lnc240,Gm31453 and Cpa5 did not affect spermatogenesis and fertility in mice.lncRNA play a fine-tuning role in the expression of relevant target genes at the molecular level.Lnc RNA deletion may have a protective effect on spermatogenesis under adverse stress.