Research On Alternative Splicing Of Ovarian Transcriptome In Xiang Pig At Estrus Stage

Pig is an important economic animal in the world.In addition to providing highquality protein and fur for humans,pigs are also ideal experimental animals in human medical research because of their physiological characteristics similar to humans.Ovary is an important reproductive organ of female mammals.In each estrus cycle,the ovaries undergo a series of morphological,hormonal and biochemical changes,which directly affect and/or determine the litter size of female animals.The function of the ovary is a complex biological process involving the transcriptional regulation of a large number of genes.Alternative splicing(AS)is an important mechanism for regulating gene expression,which regulates a variety of biological processes in higher organisms.The Chinese native Xiang pigs have many unique biological characteristics,which reach sexual maturity earlier,and their estrus behavior is not as obvious as other European pig breeds.By studying the effect of AS on the gene expression of Xiang pig ovary at estrus stage,the potential AS transcripts and signaling pathways regulating ovarian function in Xiang pigs were further revealed,so as to provide new clues and new theoretical hypotheses for exploring the molecular mechanism of ovarian function.The RNA-Seq method was used to analyze genome-wide transcripts and AS events of ovary tissues from Xiang pigs,identify AS events at estrus and diestrus stage,and compare AS patterns.At the same time,the gene expression and AS of the circadian rhythm and clathrin-mediated endocytosis(CME)related gene,which play an important role in ovarian function,were analyzed in depth.The main research results are as follows:1.Identification and functional study of AS events in the ovarian transcriptome of Xiang pigs at estrus stage.The genome-wide analysis of AS events was performed by RNA-seq method in Xiang pig ovaries at diestrous and estrous stage.Using ASprofile program,we analyzed twelve basic AS events in Xiang pig ovaries and identified 68,775 AS events in 15,142 genes from diestrous ovaries and 69,493 AS events in 15,291 genes from the estrous ovaries with average 4.54 splicing events.94.4-95.5% of expressed genes underwent alternative splicing in these tissues.The frequencies of AS events were similar to each other at diestrous and estrous.Transcription start site(TSS)was the predominant type of AS events,followed by transcription terminal site(TTS),and skipped exon(SKIP).The remaining type of AS events,e.g.,intron retention(IR)and alternative exon ends(AE),showed the lower frequencies.Further comparison analysis of gene expression indicated that 4,433 genes had at least one splice variant differentially expressed during estrous,whereas only 2,382 of them were differentially expressed at gene level by Limma,DESeq2 and r MATs softwares.Numerous genes involved in gonad development and hormone metabolism were differentially regulated through AS.Twelve genes with different types of alternatively splicing were validated by using RT-PCR method.The GO annotation and KEGG pathway analysis clearly revealed that a lot of DEGs(differentially expressed genes)and DSGs(differentially spliced genes)were involved in follicular development and ovarian steroid biosynthesis.A large number of DSGs,although not differentially expressed,were enriched in circadian rhythm and several signaling pathways.These pathways potentially regulated the female animal reproductive function in gene and/or AS level.Our results suggested that alternative splicing play an essential role in regulation of gene expression in female pigs during estrous.Numerous genes involved in gonad development,hormone metabolism,circadian rhythm were differentially regulated through alternative splicing.2.Research on the expression and AS of ovarian circadian rhythm-related genes of Xiang pigs at estrus stageThe transcriptome sequencing technology was used to analyze the expression of circadian rhythm-related genes and the alternative splicing of transcripts in Xiang pigs ovaries at estrus and diestrus stage.Among the 90 rhythm-related genes,81 were detected in the diestrous ovaries,and 83 were detected in the estrous ovaries.Compared with diestrous ovaries,there were more genes with medium expression levels(10-100 CPM)in estrous ovaries,and fewer genes with high expression levels (> 100 CPM)and low expression levels(0.1-10 CPM).There were 33 rhythm genes differentially expressed between estrus and diestrus stage.From the 34 circadian rhythm gene transcripts,44 differential splicing events were identified,including 3 alternative 5’ splice site(A5SS)events,12 retened intron(RI)events,14 skipped exon(SE)events and 15 mutually exclusive exons(MXE)events.Twenty genes including the core clock components aryl hydrocarbon receptor nuclear translocator like(ARNTL)and cryptochrome circadian regulator 1(CRY1)were differentially regulated only at the AS level,and 14 genes including period circadian regulator 1(PER1)and clock circadian regulator(CLOCK)were both differentially regulated at the expression and AS levels.It showed that the estrus cycle promoted the time change of the biological clock in the ovary tissue,and the peripheral circadian clock oscillator played a vital role in synchronizing the local physiological process.3.Research on the expression and AS of CME-related genes in the ovaries of Xiang pigs at estrus stageThe transcriptome sequencing technology was used to analyze the expression of CME-related genes and the alternative splicing of transcripts in Congjiang Xiang pigs ovaries at estrus stage.Transcriptome changes at diestrus stage were used as controls.As a result,8 CME genes were detected in the ovaries of Congjiang Xiang pigs,including DAB adaptor protein 2(DAB2),adaptor related protein complex 2 subunitμ1(AP2M1),clathrin light chain B(CLTB),clathrin light chain A(CLTA),dynamin-1(DNM1),myosin Ⅵ(MYO6),dynamin-2(DNM2)and clathrin heavy chain(CLTC).Among them,the differentially expressed genes were DAB2,AP2M1 and CLTB.Except for CLTB,the other 7 genes produced different alternative splicing events between estrous and diestrous ovaries,and the 7 transcripts produced by these alternative splicing events were the new transcripts not included in the Ensembl database up to now.It showed that alternative splicing was an important regulation mode of ovary in Congjiang Xiang pigs at estrus stage,and a specific network of protein interaction was formed by producing new transcripts and proteins during CME.The results of this study indicated that the internalization of gap junction was regulated by the expression of CME-related genes and splicing of transcripts at transcription and post-transcription levels,thus regulating the ovarian function of Congjiang Xiang pig.In summary,this study used the next-generation sequencing technology to sequence the ovarian transcriptome of the Xiang pigs,and constructed the AS map of the ovarian transcriptome of the estrus stage.Pathways that can identify AS events and transcript isoforms that play an important role in ovarian function of Xiang pigs were set.These study contents are helpful to improve the understanding of post-transcriptional regulation of porcine ovarian function,and it is of great significance to further analyze the molecular regulation mechanism of porcine ovarian function.