Leptin-related LncRNA Screening In Ovary Of Sheep And Its Analysis In Seasonal Oestrus

Seasonal reproduction of sheep is a protective behavior of sheep to adapt to the environment.Sheep do not show estrus in non-breeding season,ovulation will not occur,resulting in a female sheep can only give birth to one fetus a year,and the lamb birth is also seasonal,which is the main restrictive factor affecting the efficient reproduction of sheep.Seasonal breeding sheep can also show oestrus in the absence of good nutrition.Leptin is a pleiotropic cytokine that plays an important role in the correlation between nutrient levels and reproductive processes.LncRNA is a transcription with over 200 bases and has no protein coding capacity,which can play an important regulatory role in many biological processes through various mechanisms,including transcriptional regulation,posttranscriptional control and epigenetic process.Therefore,this study screened lncRNA and leptin-related lncRNA from sheep ovaries in different reproductive states,providing theoretical basis for studying the molecular mechanism of lncRNA in the regulation of seasonal reproduction.Meanwhile,by combining the expression analysis of reproductive hormone receptor in ovary of sheep with different reproductive states and the detection of reproductive hormone concentration in serum samples,the physiological state of ovary was further understood,providing theoretical basis for the accurate analysis of the role of leptinrelated lncRNA in the regulation of seasonal reproduction of sheep.Objective: In this study,the ovaries of seasonal oestrus and perennial oestrus sheep in different reproductive states were studied to understand the physiological state of ovaries of seasonal oestrus sheep,screen the different expressions of lncRNA and mRNA related to seasonal oestrus,and explore the regulatory role of leptin-related lncRNA in the seasonal reproduction of sheep.(1)Explore the correlation between leptin long receptor expression in hypothalamus,uterus and ovary during sheep estrus.(2)Collect sheep ovary tissues in different reproductive states accurately,construct RNA sequencing library of sheep ovary and conduct sequencing analysis,providing data resources for the analysis of seasonal reproductive transcriptome.(3)Different expressed lncRNA and mRNA and leptin-related lncRNA were screened for seasonal estrus,and their regulation in seasonal estrus was analyzed.(4)Explore the differences in the expression of differentially expressed gene FSHR in the ovary tissues of Chinese merino sheep and Hu sheep,and to analyze the correlation between leptin and FSHR expression through the granulosa cells which specifically expressed FSHR.This study provides theoretical basis for systematically describing the regulatory role of lncRNA in seasonal reproduction and efficient breeding of sheep.Methods:(1)Using the hypothalamus,uterus,ovarian tissue from sheep of different estrus period,extract total RNA and reverse transcriptase,using real-time fluorescent quantitative PCR technique to detect the expression of leptin long receptor of the samples,analysis of leptin long receptor expression changes in different sheep estrous stages.(2)Collect ovary samples of perennial oestrous Hu sheep and seasonal oestrous Chinese merino in different reproductive states,extract total RNA to construct RNA sequencing library,carry out RNA sequencing through Illumina Hiseq 2500 platform,and splicing and assembling transcripts.(3)Using CNCI,CPC,Pfam Scan and PhyloCSF to analyze the coding potential of the transcripts,differential expression of lncRNA and leptin-related lncRNAs with seasonal reproduction were screened and target genes were predicted,GO analysis,KEGG analysis and lncRNA-mRNA combination analysis were carried out to analyze the regulatory role of lncRNA in seasonal reproduction.(4)Using ovarian samples with different reproductive states,the role of differentially expressed gene FSHR in the regulation of seasonal estrus was analyzed,and the relationship between leptin and its related lncRNA in the regulation of seasonal estrus and the regulation of differentially expressed gene FSHR was analyzed in combination with the granulosa cells experiment.Results:(1)Compared with the hypothalamus,the expression of leptin long receptor in ovary and uterus of Chinese merino was lower during the estrus,dioestrus and proestrus,while the latter stage of estrus was significantly higher than that of the hypothalamus.The expression trend of leptin long receptor in the same tissue of the two species of sheep was similar.(2)Accurate collected Chinese merino seasonal oestrus and perennial oestrus sheep ovarian tissue with different reproductive states,anestrus ovarian surface without corpus luteum and ovulation,the expression of ovarian PR and LHR and corpus luteum formation gene show that the gene expression level of anestrus ovarian similar to the breeding season of Chinese merino sheep and Hu sheep in estrus.Six specific RNA-seq libraries were constructed using ovarian samples,and a total of 571,389,014 original data were obtained,and 555,097,302 purified sequences,a total of 83.27 Gb data were obtained.The proportion of pure data in each library was greater than 96.74%.After comparing with the sheep genome,the mapping rate was more than 79.18%.The results of library sequencing were highly reliable and could be used in subsequent analysis.(3)1473 novel lncRNAs were screened out,and 20 lncRNAs and 152 mRNA were detected in ovary compared anestrus with other reproductive states.The total number of co-expression target genes obtained from lncRNA analysis was 99,accounting for 65.1% of the differentially expressed mRNA,indicated that the reliability of the RNA sequencing data was high.One lncRNA targeting leptin gene and nine lncRNAs related to leptin receptor gene were screened from RNA sequencing data.(4)In autumn,there was no difference in the expression of FSHR and ER in the ovarian tissue of the two species of sheep with the same physiological state.In the spring,the expressions of ER?,ER?,and FSHR were significantly higher than that of Hu sheep ovaries in estrus and dioestrus,but the secretion trend of E2 and FSH in serum was similar to that of Hu sheep.The expressions of ER?,ER? and FSHR of Chinese merino ovary were higher in spring than in autumn,while the expressions of ER?,ER? and FSHR in Hu sheep dioestrus ovary were significantly lower in spring than in autumn.The addition of low concentration of leptin can promote the expression of FSHR,while high concentration tends to inhibit,and the addition of leptin can promote the expression of luteinization related gene in granular cells.Conclusion:(1)By analyzing and comparing the expression changes of leptin long receptor in different tissues of sheep with different estrus stages,it was found that the expression changes of leptin long receptor were correlated to sheep estrus.(2)No corpus luteum and ovulation site on ovarian surface,gene expression analysis and correlation analysis of sequencing data showed that the ovarian in the stage of anestrus had highest correlation with the estrus of Hu sheep ovary in spring.(3)Different expressions of lncRNA and mRNA and leptin-related lncRNA were screened out,and had analyzed the biological functions involved in the regulation of seasonal estrus in sheep and the regulation network of lncRNA-mRNA.(4)To some extent,Leptin promotes FSHR expression and luteal formation.