The Preliminary Function Study And Screening Of Spermatogenesis And Sperm Function Related LncRNA In Mice

Background:Long noncoding RNAs(lncRNAs)are a class of non-coding transcripts which are longer than 200 nt in length.They are involved in epigenetic regulation,cancer,immunity,reproduction and and other biological processes.Lnc RNAs are mainly concentrated in the brain and testis tissues of primates from an evolutionary point of view.Further high-throughput transcriptome sequencing revealed that the expression of lncRNAs gradually increased with the development of spermatogenesis.A key testis-specific lncRNA was knocked out in Drosophila,which results in reduced sperm count or even infertility.This results indicate that lncRNAs play a vital role in spermatogenesis.Reseach suggests that there are also a large number of m RNA and small non-coding RNA in mature sperm,and have a certain effect on sperm function.However,the effect of mammalian lncRNA on spermatogenesis and its existence and function in mature spermatozoa are still blank.The purpose of this study is to screen and study the role of testis-specific lncRNA in spermatogenesis and sperm function by using high-throughput sequencing technology.This study will be of great significance to clarify the fine mechanism of spermatogenesis and male infertility.Method:Forty-five mice(10-12 weeks old)were randomly divided into three groups to provide three pools of sperm samples from 15 mice each.The testes of two mice were randomly were arranged into two biological replicates of testes samples.After collecting three groups of sperm samples and two biological replicates of testes samples.Total RNA was extracted and tested for purity and quality.These samples were named as M-sperm-1,M-sperm-2,M-sperm-3,M-testes-1 and M-testes-2,respectively.The expression characteristics of lncRNA in mouse sperm were obtained by high-throughput transcriptome sequencing.The differentially expressed lncRNA genes were screened by sequencing data of sperm,testes and round spermatids in the GEO database.After GO analysis(Gene Ontology,gene ontology)and lncRNA target-gene analysis were used to predict the lncRNA related to spermatogenesis and sperm function,lncRNA was filtered by tissue specificity or conservative analysis.Testis-specific lncRNA was obtained by RT-PCR and q PCR verification,and further expression study was carried out.Lentivirus overexpression vector and gene knockout model were used to preliminarily explore the function of candidate lncRNA.Results:1.The high purity RNA of mouse sperm was obtained in this study.2.The lncRNA expression profile in mouse sperm has been systematically investigated.From the view of length distribution,we found that the length of most lncRNAs ranged from 500 bp to 2000 bp.As for distribution of expression,the lncRNA expression level of sperm was lower than m RNA.43979 lncRNAs of sperm were differential expression,among which 12778 lncRNAs were up-regulated and 31201 lncRNA were down-regulated,compared with the testes.37164 lncRNAs of sperm were differential expression,among which 15148 lncRNAs were up-regulated and 22016 lncRNAs were down-regulated,compared with the round spermtids.The GO analysis divides target-m RNA into 3 categories: biological processes,cell components,and molecular functions.In comparison of testes,the most significant biological processes of sperm involved in the biological process were cellular macromolecule metabolic process,reproduction,sexual reproduction,male gamete generation,primary metabolism and spermatogenesis.In comparison of spermatids,the most significant biological processes of sperm involved in the biological process were cell metabolism process,cell metabolism process,reproduction,sexual reproduction,male gamete production,primary metabolism process,etc.KEGG analysis showed that the differentially expressed target-m RNA of significant enrichment pathways between sperm and testes were related to endocytosis,micro RNAs in cancer,RNA transport,adhesion junction,and so on.The pathways of target-m RNA enrichment between sperm and spermatids were mainly related to endocytosis,RNA transport and Spliceosome.3.Applying for bioinformatics screening,108 testis-specific lncRNAs related to spermatogenesis and sperm function were obtained.By using RT-PCR and q PCR,we obtained 8 testis-specific lncRNA related to spermatogenesis and sperm function.4.Through the analysis of the gene location and lncRNA target-gene,this study selected the antisense transcript of the sperm cell calcium binding protein gene Cabs1,the conserved lncRNA lnc5 and the testis-specific lncRNA lnc49 which will be considered as the research objects to the further exploration their specific function.In this study,we constructed two stable cell lines respectively overexpressed lnc5 and Cabs1 genes.Furmore,we discussed whether lnc5 regulates Cabs1 gene expression.The results showed that lnc5 did not affect the RNA level and protein level of Cabs1.In this study,lnc5 and lnc49 gene knockout models were constructed by CRISP/Cas9 technology and F1 positive heterozygotes were obtained.Conclusion:1.This study confirmed that the fragment of the transcripts of the mouse sperm,a large number of intact lncRNA were found,and the expression profiles of sperm lncRNA were systematically studied and elucidated.2.There were significant difference in the expression of lncRNA among mature sperm,round spermatids and testes.Function enrichment analysis of differentially expressed lncRNA genes showed that they affected a variety of biological processes related to spermatogenesis and sperm function.3.According to bioinformatics prediction,testis-specificity analysis by using RT-PCR and q PCR showed that eight testis-specific lncRNAs related to spermatogenesis and sperm function were obtained.This result provided a basis for further study of the biological functions of testis-specific lncRNA.4.In this study,lnc5 overexpression vector,lnc49 knockout model and lnc5 knockout model were constructed.Preliminary results showed that the transcription and translation level of Cabs1 gene was in an independent manner.At present,the phenotypic analysis of knockout models is in progress.We will further study the regulatory relationship between lnc5 and Cabs1,as well as the role of lnc5 and lnc49 in spermatogenesis and sperm function in vivo and in vitro.