The Analysis Of Effect Of High-and Low-Dietary Intake During Luteal Phase On Follicular Development Of Hu-sheep Using Expression Profiling

Nutrition is one of the important environmental factors that affect the reproductive performance of sheep.Short-term supplement feeding during luteal phase promotes follicular development and increases the ovulation rate of sheep.But short-term supplement feeding in follicular phase does not show these kinds of effect.Previous study in our lab also indicated this facilitated effect of short-term supplement in luteal phase.However,the molecular mechanism between nutrition level and follicular development is currently unknown.Therefore,a better understanding of this process at molecular level may result in new practices to improve sheep breeding,which is important in stockbreeding development.It also helps to discover the molecular mechanism responsible for nutrition regulation on reproductive performance of sheep.In order to investigate the gene expressions and their relation with follicular development,16 ewes from high-dietary intake group(H;1.5M;n=8)and low-dietary intake group(L;0.5M;n=8)were slaughtered.RNA was extracted from H and L group granulosa cells which were scraped from follicles>2.5 mm in diameter.Expression profile sequencing was performed on RNA from these two groups using RNA-seq method,and 170 differentially expressed genes(DEGs)were found according to the sequencing data.To understand the expression profile and the function of genes,we performed GO analysis,KEGG analysis and clustering analysis.1.Illumina expression profile sequencing was performed on RNA extracted from H and L group granulosa cells.We obtained approximately 2.4 Gb clean data,including 49,139,471 clean reads.The reads were mapped to the sheep reference genome database,4,259,559(91.81%)and 20,777,451(91.47%)reads from L and H group matched genomic locations respectively.We obtained 18,468 genes expressed in total library.The data that covered most of the transcriptome sequences were reliable.Among these genes,16,523 genes were expressed in both samples.Filtered with FDR<0.05,170 genes showed a significance of expression difference,including 140 up-regulated genes and 30 down-regulated genes.This sequencing data provided important genetic information resources for investigating the molecular mechanisms of sheep follicular development.2.To classify the function of 18468 detected genes,GO annotation was performed.In total,15,278 genes were categorized into 60 functional groups.We annotated 6361 genes to 312 KEGG pathways.After GO analysis and KEGG pathway enrichment,170 DEGs were categorized into 47 GO functional groups and were aligned into 120 KEGG pathways.We observed some reproductive regulation pathways,such as "Metabolism of xenobiotics by cytochrome","Oocyte meiosis","GnRH signaling pathway","Progesterone-mediated oocyte maturation","steroid hormone biosynthesis",and etc.Combining GO functional enrichment and KEGG pathway analysis,we found 5 up-regulated genes,PTGS2,UCP2,STAR,ILIA and IL1B,involved in follicular development.3.According to the similarity of 170 DEGs expression profiles,hierarchical clustering assigned them into 10 expression cluster groups,subcluster 1 to subcluster 10.Their clusterings and expression pattern were described in detail.We found that PTGS2,UCP2 and STAR were clustered in subcluster 1.Additionally,IL1A and IL1B were clustered in subcluster 3.It indicated that these two groups DEGs had similarities on expression patterns respectively and were associated with follicular development.These 5 genes were worthy of further study.We analyzed the expressions of the 5 genes according to FPKM and qRT-PCR data.Two results were consistent.All these 5 genes showed higher expression levels in high-dietary intake group during luteal phase(H group).The results suggested that PTGS2,UCP2,STAR,ILIA and IL1B might be important candidate genes which promoted follicular development.This study provided important genetic information resources for investigating the differentially expressed genes in granulosa cells from sheep fed by different nutrient levels and the molecular mechanisms of sheep follicular development.