Exploration Of Male Reproduction-related Genes And Their Regulation Mechanisms In Pig(Sus Scrofa) Using Cross-species RNA-seq Data

Pig（Sus scrofa）is not only an important economic animal,but also an excellent biomedical model animal.Male reproduction is a complex process that involves cell fate decisions and specialized cell divisions,which especially required precise coordination of gene expression in response to both intrinsic and extrinsic signals.And the emergence of high-throughput RNA sequencing（RNA-seq）has led to a deep and detailed understanding of mammalian male reproductive regulatory genes.However,a majority of the current studies exploring mammalian reproduction using high-throughput RNA-seq techniques are focused on common model animals such as mice,and the genes related to pig male reproduction and their transcriptional characteristics and molecular regulatory mechanisms remain yet uncharacterized and need to be systematically explore.Furthermore,given that many genes related to male reproduction are usually expressed specifically in the testis,this study intend to start from the perspective of testis-specific genes（TSGs）.Specifically,this study,using public RNA-seq data of five mammals（pig,cattle,sheep,human and mouse）,in-silico predicted porcine transcription factor binding sites and porcine protein interaction data,for the first time to systematically study the transcriptional characteristics of porcine TSGs and the molecular mechanism of their expression.The main results of this study are as follows:1.Dynamic gene expression patterns revealed pig reproductive traits related genesBy analyzing the RNA-seq data of pig various tissues,this study found that the transcription of protein-coding genes was more frequent in testis than that in other tissues.And much more highly expressed genes（log₂FPKM≥4）were present in testis than in other tissues.Combined with tissue specificity index,GO and GSEA analysis,1,210protein-coding genes were identified with specific expression pattern in pig testis,and these genes were significantly enriched in functions and pathways related to male reproductive（i.e.,spermatogenesis,germ cell development,spermatid differentiation,and others）.2.Characterization of unique or conserved during evolution TSGs in the pig using cross species comparisonUsing cross-species comparison,porcine TSGs with different expression conservation were uncovered in this study:195 TSGs with 87 TSGs with low expression conservation（LCTSGs,exclusively to pig）,113 TSGs with moderate expression conservation（MCTSGs,present in pig,cattle and sheep）and high expression conservation（HCTSGs,shared by pig,cattle,sheep,human and mouse）.Among them,compared with LCTSGs and MCTSGs,HCTSGs had significantly higher expression abundance and tissue specificity.And there were differences in the functions of these three gene sets,HCTSGs are more likely to be enriched to male reproduction-related functions.3.Exploration of the molecular mechanism that regulates the transcriptional of porcine TSGsFurther analysis of the results suggested that post-transcriptional regulation mechanism,such as alternative splicing（AS）,was also involved in the regulation of porcine TSGs.Changes in A5（alternative 5’splice sites）,AF（alternative first exons）,and SE（exon skipping）events were the predominant features of TSGs splicing regulation in pig,and the high expression of TSGs was mainly the result of the high expression of one of their isoforms.Additionally,transcriptome analysis also showed that most of the transcription factors（TFs）have a potentially negative regulatory effect on TSGs expression.TCF7L1 and THRB might be key TFs in the regulation of porcine TSGs.Finally,this study constructed a porcine TSGs regulatory network,via which this study found that the degree centrality,betweenness centrality and closeness centrality were significantly lower in TSGs than in NTSGs.At the same time,this study revealed 41 hub TSGs in pig that directly interacted with the majority of other testis-specific candidate genes and might be involved in regulatory cascades related to reproduction.Overall,this study deepens our understanding of the male reproductive regulation mechanism in pig from the perspective of TSG transcription and provide a scientific basis for improving pig reproductive performance.