| Follicles are the core functional units in the development of mammalian oocytes.The process of oocyte growth and maturation takes place within the follicles.Granulosa cells are one of the important cells that make up follicles.During the formation and development of follicles,granulosa cells interact with oocytes,thus influencing the development,maturation and atresis of follicles.Using FSH to treat lambs aged 4-10 weeks can obtain the number of developing follicles about more 10 times than adult sheep.Using lambs as donors can not only provide a large amount of oocyte resources for embryo in vitro production,but also greatly shorten the generation interval and accelerate the propagation of improved breeds.It also provides a good experimental model for further exploration of follicular development in sheep.This study Chinese merino(xinjiang type reclamation)adult ewes naturally big follicles(ELF),small follicle(ESF)and hormone treatment after large follicles(EFF),and the lamb hormone treatment(LSF)before and after treatment(LFF)of ovarian granular cells(GCs)as materials,has carried on the depth of the small RNA sequencing respectively,transcriptome sequencing and proteomic sequencing analysis.The differentially expressed miRNA,RNA and protein were analyzed jointly,and the function of specific miRNA and its predicted target gene was verified on the cultured sheep GCs.The purpose of this study was to reveal the potential molecular regulatory mechanism of granulosa cells in sheep ovary through multi-group analysis,so as to provide theoretical basis and technical support for the further study of follicular development and reproductive system development and regulation of sheep in order to make better use of in vitro embryo resources of sheep or lambs.The main research results are as follows:1.Sequencing of small RNA of granulosa cells in adult sheep and lamb at different follicular development stagesThe five sample groups obtained the largest amount of small RNA with a length of 22 nucleotides,accounting for about 30% of all small RNA.A total of 146 known mirnas were identified,among which the EFF group had the most(145)and the LSF group had the least(120).Compared between each group,the least differentially expressed mirnas were observed between LFF and LSF(9),and the number of differentially expressed mirnas between the other groups was 20-101.For example,there were only 9 mirnas(|log2(FC)| > 1 and p< 0.05)between the two groups of lambs and adult sheep after hormone treatment(LFF vs EFF),and five miRNAs were down-regulated and four miRNAs were up-regulated in the lambs.Analysis of the GO and KEGG pathways of the predicted target genes of miRNAs showed that they were mainly enriched in metabolism,cancer pathway,PI3K-Akt pathway,MAPK pathway and immune-related pathway.After FSH stimulation,miRNAs such as oar-mir-21 and oar-mir-143 were down-regulated in both ewes and lambs,indicating that FSH has an inhibitory effect on these miRNAs and may affect follicular development through its target genes.2.GCs transcriptome sequencing of lambs and adult ewes at different follicular development stagesA large number of differentially expressed genes were also screened for comparison between the two groups.Taking LFF vs EFF as an example,a total of 311 genes were differentially expressed,among which 136 were up-regulated and 175 were down-regulated.It has been reported that genes related to follicular development or development,such as STAR and CYP19A1,were up-regulated in lambs,while EFEMP1,PTX3,ADM and OXT were down-regulated.These differentially expressed genes were mainly concentrated in the extracellular region and extracellular matrix.It mainly involves the cell cycle,DNA repair,steroidogenesis and PI3K-Akt pathways.Nine genes from the two groups were selected for qPCR,and the results were consistent with the trend of sequencing results.In order to screen out the mirnas affected by FSH trentment and their target genes,we compared the differences of mirnas between adult sheep and lambs after hormone treatment,before and after lamb hormone treatment and after adult sheep hormone treatment,and found that mir-21,mir-17-5p and mir-29b-3p were common differences of mirnas.IPA software was used to predict and analyze the differentially expressed genes in the three mirnas and the corresponding groups,and eight differentially expressed genes were identified: CDK6,PIK3R1,MAP3K1,EGR3,TNF,STAT3,PTEN and BCL2.The functions of these genes are related to angiogenesis,granulosa cell apoptosis and ovary ovulation,suggesting that these genes may regulate the expression of target genes by miRNA under the action of FSH trentment and participate in the follicular development process of sheep.3.Proteomic analysis of ovarian granulosa cells after lamb and adult ewe treatmentThe iTRAQ proteomic analysis of GCs from steroid-treated lambs and adult sheep identified a total of 574 differentially expressed proteins,of which 338 were up-regulated and 236 down-regulated.The differentially expressed proteins concentrate on biological processes such as biosynthesis,nitrogen metabolism and stress response.There may be interaction among 50 differentially expressed proteins related to follicular development and GCs proliferation.Five differentially expressed proteins were selected for qPCR and Western Blot validation,and the results were consistent with iTRAQ results.4.Molecular regulation analysis of miRNA on PIK3R1 gene expressionCompared with adult sheep,PIK3R1 expression was significantly up-regulated in GCs of lambs,and its 3-UTR was also a predicted target of miR-21.Therefore,we conducted preliminary verification of its expression regulation at the cell level in vitro.The luciferase reporter vector of PIK3R1 gene was constructed and co-transfected with miR-21 into 293 T cells,and it was found that the latter could significantly inhibit the Dul-luciferase activity.miR--21 was transfected into sheep for GC,and the expression of PIK3R1 gene was detected by qPCR and Western Blot.The results showed that mir-21 not only significantly inhibited the transcription level and protein expression level of PIK3R1,but also promoted cell proliferation,indicating that mir-21 may degrade target genes by binding to specific sites in the 3'utr region of PIK3R1 gene.In summary,the conclusions are as follows: there are significant differences in the expression characteristics of miRNA,transcriptome and proteome in follicular granulosa cells of adult ewes and lambs at different development stages and after hormone treatment.FSH may regulate the development of ovine follicles through the expression changes of differential miRNA and its target genes in granulosa cells,which involves the regulation of PIK3R1 gene expression by miR-21 in sheep granulosa cells. |
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