| Male sheep reproductive performance is mainly reflected in breeding capacity and semen quality,and semen quality is closely related to testis tissue structure.Testis development and spermatogenesis are important factors affecting male sheep reproductive ability.Small Tail Han Sheep are an excellent local breed in China,which are famous for their high fecundity.Small Tail Han Sheep are also a female breed widely used in meat sheep industry and play an important role in the development of meat sheep industrialization in China.In this research,male Small Tail Han Sheep were taken as research object.1.We studied reproductive performance of Small Tail Han Sheep and selected 2-,6-,and 12-month-old Small Tail Han Sheep testes to observe their histological structure using H.E.staining method.2.Based on the differences of histological structure and spermatogenesis process,mRNA library and sRNA library of 2-,6-,and 12-month-old Small Tail Han Sheep testes were constructed;mRNA,miRNA and miRNA-mRNA expression profiles were analyzed using RNA-seq technology and bioinformatics methods;miR-Targets regulatory net-works were constructed to select the key miRNAs and its target mRNAs in testis development and spermatogenesis.3.The sequencing data were validated using qRT-PCR;targeted regulation of key miRNAs and its target mRNAs in testis development and spermatogenesis were validated by double luciferase reporter gene detection system.The purpose of this research was to provide theoretical basis for revealing the molecular regulation mechanism in testis development and spermatogenesis of Small Tail Han Sheep and finding out the molecular markers affecting their excellent reproductive performance.The main results of this research are as follows: 1.Investigation of Small Tail Han Sheep reproductive performance: The results showed that Small Tail Han Sheep has the characteristic of sexual precocity,male lambs develop sexual behaviour at 75-88 days of age,and sexual maturity can be achieved at 6 months old;Small Tail Han Sheep testes development is positively related to testosterone secretion,and the semen of male Small Tail Han Sheep has the advantages of high ejaculation,high density,high sperm motility,long sperm survival time in vitro and good semen quality,which can be used for perennial breeding.There are significant differences in the diameter of seminiferous tubules and the number of spermatogenic cells of 2-,6-,and 12-month-old Small Tail Han Sheep,which can be used for transcriptome diference analysis.2.mRNA expression profile analysis of Small Tail Han Sheep testes at different months of age: The mRNA libraries of 2-,6-,and 12-month-old Small Tail Han Sheep testes were successfully constructed,630 differentially expressed genes(DEGs)were obtained from 2-vs 6-month-old testes,which contained 470 up-regulated and 160 down-regulated genes;322 DEGs were obtained from 6-vs 12-month-old testes,which contained 218 up-regulated and 104 down-regulated genes;102 DEGs were obtained from 6-vs 12-month-old testes,which contained 82 up-regulated and 20 downregulated genes.GO and pathway analysis showed that the DEGs in 2-vs 6-month-old testes were significantly enriched in 191 biological processes(such as male courtship behavior,steroid catabolic process and spermatogenesis)and 17 signal pathways(such as amino sugar and nucleotide sugar metabolism,nucleotide excision repair and AMPK signaling pathway);the DEGs in 6-vs 12-month-old testes were significantly enriched in 311 biological processes(such as sexual reproduction,developmental programmed cell death and sperm capacitation)and 8 signal pathways(such as ribosome,other glycan degradation,riboflavin metabolism).The total of 955 DEGs in 2-vs 6-monthold,6-vs 12-month-old,and 2-vs12-month-old testes was categorized into 8 profiles by series cluster.The eight profiles were classified into 4 model profiles and 4 coexpression networks were constructed based on the DEGs in these model profiles.13 genes(CA3,APOH,MYOC,CATSPER4,SYT6,SERPINA10,DAZL,ADIPOR2,RAB13,CEP41,SPAG4,ODF1 and FRG1)were randomly selected to validate the sequencing data using qRT-PCR method,and the qRT-PCR results were consistent with RNA-seq data,which proved the accuracy of mRNA sequencing data.3.miRNA expression profile of Small Tail Han Sheep testes at different months of age: miRNA libraries of 2-,6-and 12-month-old Small Tail Han sheep testes were successfully constructed,5,1,and 4 differentially expressed sheep known miRNAs and 132,105,and 124 differentially expressed novel miRNAs were identified in 2-vs 6-,6-vs 12-,and 2-vs 12-month-old testes.6 differentially expressed sheep known miRNAs(oar-miR-379-5p,oar-miR-665-3p,oar-miR-200 b,oar-miR-409-3p,oar-miR-495-3p and oar-miR-200c)and 6 differentially expressed novel miRNAs(has-miR-3592-5p,has-miR-6820-3p,has-miR-3615,mmu-miR-1957 b,mmu-miR-182-3p and mmu-miR-1929-3p)were selected to validate the sequencing data using qRT-PCR method,and the qRT-PCR results were consistent with RNA-seq data,which proved the accuracy of miRNA sequencing data.4.Target gene prediction of differentially expressed miRNA: Combining the results of miRNA and mRNA to predict the target genes of differentially expressed miRNAs,68,1,and 9 target genes of differentially expressed known miRNAs and 363,168,and 29 target genes of differentially expressed novel miRNAs were obtained in 2-vs 6-,6-vs 12-,and 2-vs 12-month-old testes,respectively.GO and pathway analysis showed: in 2-vs 6-month-old testes,the target genes of differentially expressed known miRNAs were involved in 100 biological processes(such as apoptotic process,meiosis I,and acrosome reaction)and 11 signal pathways(such as Hedgehog signal pathway and Wnt signal pathway);in 6-vs 12-month-old,the target genes of differentially expressed known miRNAs were involved in 4 biological processes(multicellular organism reproduction,binding of sperm to zona pellucida,single fertilization and proteolysis);in 2-vs 12-month-old,the target genes of differentially expressed known miRNAs were involved in 17 biological processes(such as adenosine metabolic process,purine nucleobase metabolic process and nucleotide metabolic process)and 4 signal pathways(Riboflavin metabolism,Glutathione metabolism,Thyroid hormone synthesis and Arachidonic acid metabolism).5.Selection and validation of key miRNAs and their target genes in testis development and spermatogenesis: 3 miR-Targets regulatory networks were constructed,based on GO and KEGG analysis,oar-miR-379-5p and its candidate target gene WNT8 A and oar-miR-665-3p and its candidate target gene CCIN were selected as the key miRNAs and target genes in testis development and spermatogenesisy.The results of double luciferase reporter gene detection system demonstrated that WNT8 A is the target gene of oar-miR-379-5p and CCIN may not be the target gene of oar-miR-665-3p. |
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