Preliminary Study On The Biological Function Of PiRNAs And Piwill Gene During Spermatogenesis In Poultry

Germ cells are responsible for the generation of genetic information,and their genomic characteristics,expression and regulation patterns are essential for individual and species maintenance.As half of the genetic information carrier spermatozoa has not been given enough attention in poultry.But in recent years,the problem of continuous decline in chicken fecundity has always plagued the development of the industry.5?12%of the chickens in the local breed are eliminated due to spermatogenesis failure.In modern commercial breeds,the sperm quality of roosters causes the seedling rate to decline year by year.In addition,the sperm-mediated bacterial microbes(Salmonella pullorum,Campylobacter jejuni,etc.)and viral diseases(avian leukemia,EDS-76,etc.)increased year by year,which plagued the industry for many years,"two white"(white diarrhea and leukemia)not yet resolved.Therefore,the study of chicken spermatogenesis and its impact mechanism has become one of the urgent problems.piRNA(Piwi-interacting RNA)is a small RNA of about 30 nt length,and PIWI protein family members play a regulatory role.Gene silencing caused by PIWI/piRNAs complex can regulate the proliferation,differentiation and spermatogenesis of germ stem cells in mice such as mice,Drosophila,zebrafish,etc.However,there are few reports on PIWI/piRNAs in poultry and the function has not yet been clarified.In this study,we analysis the function of Piwi and piRNAs in spermatogenesis from several side like:the profile of small RNAs in the germ cells of different stages of spermatogenesis was analyzed,the profile of piRNAs which combine with PIWI protein,the relationship between piRNAs and target genes,the function of Piwi in meiosis and the affection of spermatogenesis to chicken after knockout Piwi.Provide the theory for biological function in poultry and the improvement of poultry fecundity,and also add new information for the study of epigenetic regulation mechanism of germ cell and spermatogenesis.In order to explore the piRNAs involved in chicken spermatogenesis and germ stem cell proliferation,the small RNAs expression profiles of different germline stem cells(PGCs,SSCs)and spermatogenic cells(Sa,Sperm)were obtained by deep sequencing.The results showed that chicken piRNAs were mainly between 31-33 nt,which were slightly longer than those of piRNAs in mice.They were mainly located in the coding gene sequence and transposon region.The results of base preference analysis showed that chicken piRNAs showed the 5'-base U,position 10 is characteristic of A(1U,10A),and the base preference of Sa cells and piRNAs in Sperm is more pronounced.In addition,a large number of piRNA clusters were predicted in Sa cells and Sperm,respectively,located in different chromosomes.We infer that chicken piRNAs were similar to mice's and have important regulatory effects on spermatogenesis.Through each cell analysis and different groups compared(PGCs vs.SSCs;Sa.Vs.Sperm),several piRNAs were enriched and acquired target genes associated with chicken spermatogenesis and germ stem cell proliferation.Five target genes:KIT,SMAD6,SCX,PGR and GGNBP2 were obtained after GO enrichment of the target gene.The piRNAs corresponding to these target genes were piR-gga-19128(KIT),piR-gga-403821(SMAD6,SCX),piR-gga-123686(PGR),piRNA-396(GGNBP2).At the same time,the signal pathway enriched by KEGG was:Melanogenesis,Notch signaling pathway,Steroid hormone biosynthesis,Cytokine-cytokine receptor interaction,Wnt signaling pathway and TGF-beta signaling pathway.To further verify the binding status of piRNAs and PIWI protein in poultry and the possible role of spermatogenesis and reproductive stem cell proliferation and differentiation in chicken,we used immunoprecipitation technique covered with deep sequencing to obtain small RNAs expression profiles of PGCs,SSCs and Sperm.Length analysis showed that piRNAs bound to PIWIL1 protein were mainly concentrated at 31-33 nt.The base preference also showed "lU-10A",and the base preference in Sperm was more obvious than that of PGCs and SSCs.The above analysis results were consistent with the previous studies,suggesting that chicken piRNAs do bind to PIWI proteins and participate in the regulation of chicken spermatogenesis and proliferation and differentiation of germ stem cells.The piRNAs of piRNA-19128,piRNA-70672,piRNA-64217,piRNA-139648,which bind to PIWI protein and participate in spermatogenesis and reproductive stem cell proliferation,were screened by venn analysis of three kinds of cells.Candidate genes were obtained by target genes annotation and GO enrichment analysis:KIT,SRC,WNT4,HMGB2.KEGG enrichment analysis obtained the following signal pathways:Steroid hormone biosynthesis,Notch signaling pathway,Melanogenesis.Based on the analysis of the above two parts of piRNAs,piRNA-19128 was further investigated by Venn analysis.Therefore,piRNA-19128 was used to validate the key piRNAs.Some reports have shown that KIT genes are involved in meiosis during spermatogenesis and play an important regulatory role.Therefore,the target gene KIT corresponding to piRNA-19128 was selected as the candidate one.To further verify the relationship between piRNA and KIT,the expression of KIT and piRNA-19128 in PGCs,SSCs,Sa and Sperm were detected by RT-qPCR.The results showed that the the higher expression of KIT but lower expression of piRNA-19128 in SSCs and Sperm(P<0.01).The expression of piRNA-19128 in PGCs and Sa was higher,but the expression level of KIT was low.We speculated that there was a relationship between piRNA-19128 and KIT.Next the expression of piRNA-19128 was detected by using piRNAs-19128 mimetic and inhibitor RT-qPCR.The results showed that the expression of piRNA-19128 increased with the increase of the transfection concentration of piRNA-19128 mimics to DF-1 cells,and the expression of KIT gene decreased.With the transfection concentration of inhibitor increased,piRNA-19128 expression decreased,KIT gene expression increased.Dual-luciferase assay showed that the fluorescence activity of mutant KIT gene was significantly higher than that of wild type(P<0.01).In summary,there was a relationship between piRNA-19128 and KIT,and piRNA-19128 inhibition of KIT transcription,and thus involved in the process of spermatogenesis in the meiosis.In order to further explore PIWI and piRNA regulate chicken spermatogenesis,we selected the key event-meiosis as a functional research platform to explore the Piwill(Piwi like-1),which encode chicken PIWI protein,KIT and piRNA-19128 expression.In this study,PGs cells were induced by retinoic acid,and the changes of Piwill mRNA and protein were detected by RT-qPCR,western blot and flow cytometry.Some research have shown that Piwill gene is the most expressed in round sperm cells,followed by spermatogonia,suggesting that Piwill gene involved in meiosis.The Stra8 was marker of meiosis which was significantly increased with the increase of induction time.Flow cytometry analysis showed that 44.9%of the cell morphology changes from haploid to diploid in 144h induced cells.Thus inferring that the cells enter the meiotic state.The expression of Piwill,KIT and piRNA-19128 increased with the increase of induction time.The PIWIL1 protein level showed that PIWIL1 protein expression increased with the increase of induction time.It is concluded that in poultry,the Piwill,KIT and piRNA-19128 were involved in the process of chicken spermatogenesis and regulate meiosis.To elucidate the function of PIWI/piRNAs complex in poultry(chicken)spermatogenesis,we used CRISPR/Cas9 technique to knock out Piwill gene in vivo and in vitro.Three knockout vectors were constructed for the third exon8 of Piwill gene.After transfection of DF-1 cells,T7E1 digestion and PCR amplification were used to identify the knockout activity at position 2.Activity vector were transfected with DF-1 cells.Fifty-eight clones were obtained by restriction dilution.The PCR amplification and sequencing.The results showed that the two cell lines were positive and one was a total loss 23 bp,another loss 8 bp.And knockout efficiency was 3.44%,which was slightly lower than mice.The expression of Piwill gene in different tissues of 5-day-old(heart,reproductive ridge)and 18-day-old(heart and testis)were detected by embryo hypocenter injection.The results showed that iin the organization of 5 days and 18 days of age,the knockout site appeared mutation.RT-qPCR results showed that the expression level of Piwill and SOX2 in experimental group was significantly lower than control group(P<0.01),indicating that knockout vector was successfully introduced into chicken embryo and play the function Feeding the F0 chickens and test the body weight and quality of sperm.The results indicated that the knockout Piwill had no effect on the body weight(P>0.05).Sperm quality test showed that the lack of Piwill gene,chicken sperm viability,activity decreased,and cloudiness is not obvious and the volume of sperm was lower.Selfing the F0 generation,we acquired F1 generation.PCR amplification of target sites and sequencing showed that Piwill mutations occurred in 10 individuals.In summary,the Piwill was involved in spermatogenesis and play an important role in poultry.CRISPR/Cas9 technology is suitable for poultry genome modification the modification effect could be inherited.