Transcriptomics-based Identification Of Functional Genes And Their Expression Regulation In Tibetan Sheep Testes

The testis development and spermatogenesis directly determine the fertility of rams.As one of the most important livestock species in the Tibetan Plateau and adjoining regions of China,Tibetan sheep has the reproductive characteristics of low fecundity and late sexual maturation.Thus,studying the regulatory mechanism of testis development and spermatogenesis of Tibetan sheep is of great practical importance to the ovine reproductive biology research.Nevertheless,the current knowledge of the molecular biology involved in the functional maintenance of testes in sheep,especially Tibetan sheep,is very limited.In this study,testes from 3-month-old(sexually immature),1-year-old(sexually mature)and 3-year-old(adult)Tibetan sheep were used as the test material for the following studies:1)Testicular m RNA,circ RNA,mi RNA expression profiling at three developmental stages was performed using RNA-seq technology and bioinformatics analysis,followed by the differential expression analysis and functional enrichment analysis;2)The ce RNA regulatory network was constructed according to the “ce RNA hypothesis” and the changing trends of circ RNAs,mi RNAs and m RNAs in expression;3)Based on transcriptome data from 3-month-old and 1-year-old testes,the potential candidate genes associated with immunological homeostasis maintenance were identified,and mi RNA-m RNA module and circ RNA-mi RNA-m RNA network enriched by these genes were analyzed.4)Primary sheep Sertoli cells were isolated and cultured to investigate the biological roles of crucial gene for spermatogenesis and its associated circ RNA-mi RNA-m RNA axis in ovine testicular cells.The major results of the study were as follows:1.There were 26084(10247 up-and 15837 down-regulated),57(27 up-and 30down-regulated)and 25535(10017 up-and 15518 down-regulated)differentially expressed m RNAs;3982(2079 up-and 1903 down-regulated),414(201 up-and 213 down-regulated)and 4060(2107 up-and 1953 down-regulated)differentially expressed circ RNAs;715(561up-and 154 down-regulated),57(46 up-and 11 down-regulated)and 790(628 up-and 162down-regulated)differentially expressed mi RNAs identified in 3-month-old vs 1-year-old,1-year-old vs 3-year-old and 3-month-old vs 3-year-old,respectively.RT-q PCR validations for randomly selected 10 m RNAs,10 circ RNAs and 12 mi RNAs showed that the transcriptome data acquired by RNA-seq were reliable.GO and KEGG analysis for differentially expressed m RNAs,source genes of differentially expressed circ RNAs,and their common genes indicated that they were all involved mainly in the biological processes associated with growth,development,reproduction,immunity,and metabolism;were all enriched significantly in reproduction-related pathways such as m TOR and TGF-beta,as well as signal pathways associated with the cellular junction or development such as tight junction and meiosis.2.Through co-expression ce RNA network analysis for circ RNAs,mi RNAs and m RNAs,the source genes of differentially expressed circ RNAs were primarily engaged in biological processes relevant to cell development and reproduction,as well as signaling pathways such as adherens junction,focal adherens,ECM-receptor interaction,regulation of actin cytoskeleton,MAPK,Wnt and TGF? pathway.On this basis,the ce RNA network for genes strongly correlated with testicular function,including spermatogenesis,germ cell development,meiosis,cell cycle and blood-testis barrier,were constructed.BOLL was predominantly expressed in post-meiotic round and elongated spermatids,and its expression was regulated by circ?029155/mi R-760-3p signaling axis via RT-q PCR,Western blot,immunofluorescence and dual-luciferase assays.3.A total of 1118 differentially expressed m RNAs associated with immunity(including873 down-regulated and 245 up-regulated m RNAs)were screened,based on the transcriptome sequencing and functional enrichment analysis data derived from pre-pubertal and post-pubertal Tibetan sheep testes.GO and KEGG enrichment analysis indicated that these genes were mainly enriched in biological processes such as immune system development,cell junction/adhesion,regulation of immune response and regulation of response to stimulus,as well as signal pathways including cell adhesion,T cell and B cell receptor signaling pathways,leukocyte transendothelial migration,chemokine signaling pathway,NF-?B signaling pathway and PI3K/Akt signaling pathway.On that basis,the potential mi RNA-m RNA network and circ RNA-m RNA-m RNA network for candidate genes that were involved in the maintenance of immune homeostasis were constructed.Furthermore,dual luciferase reporter assays verified the targeting associations between ITGB1/circ?013761/circ?014236 and mi R-29 b ad well as between ITGB1/circ?001254 and mi R-1185-3p.4.According the established ce RNA network,IGF1 gene was potentially regulated by oar-mi R-29 b and circ?026259.RT-PCR,RT-q PCR,fluorescence in situ hybridization and immunofluorescence analysis revealed their similar cell localization signatures in developmental Tibetan sheep testes that they were extensively expressed in germ cells,Leydig cells,and Sertoli cells.Subsequently,ovine testicular Sertoli cells were successfully isolated using two-step enzymatic digestion.RNase R digestion method,RT-PCR and Sanger sequencing confirmed the circular character of circ?026259 that was derived from the exons 2-5 of ovine KLHL5 gene,and the exon 2 and exon 5 were back-spliced to form the circular structure(named circ?026259 as circ KLHL5).Circ KLHL5 was shown to promote IGF1 gene expression through inhibition of oar-mi R-29 b in Sertoli cells,and thereafter induced the proliferation and suppressed the apoptosis of Seroli cells,through a series of dual-luciferase,overexpression,knockdown,CCK-8,Ed U staining and flow cytometric assays.Taken together,the transcriptome sequencing combined with relevant molecular biology methods reveal many differentially expressed genes that exhibit an age-dependent expression patterns in developmental Tibetan sheep testes are participated in germ cell development,meiosis,blood-testis barrier,immune homeostasis and other biological processes,and the expression of most of these genes may be regulated by mi RNAs and/or circ RNAs in response to the ever-developing intratesticular environmental and continuous spermatogenesis.Meanwhile,a novel circular RNA derived from ovine KLHL5 gene,circ KLHL5,is identified and confirmed to modulate IGF1 expression and cell development by targeting oar-mi R-29 b in sheep Sertoli cells.These findings fill this gap of knowledge on circ RNAs in sheep testis,enrich the available transcriptomic information of sheep,and provide a theoretical basis for understanding the molecular mechanisms regarding testicular development and spermatogenesis in sheep and other highland animals.