| Ovarian ovulation is a reproductive physiological process which is influenced by multiple factors.Ovulation rate is an important reproduction trait which determines the litter size of livestock.Therefore,it is necessary to investigate the mechanism of ovarian follicle development to improve reproduction traits.In this study,we focused on the follicle development of Shaanbei white cashmere goats using high-throughput sequencing,bioinformatics analysis,Western Blotting,cell culture,and Fluorescence activated Cell Sorting(FACS)to select key genes and micro RNAs(mi RNAs)and verify their functions that are significantly related to follicular development.The main results of this study are as follows:1.Dominant follicles(DF)and affiliate follicles(AF)were separated from 3 oestrum goats and 6 m RNA-seq,as well as 6 s RNA-seq libraries were constructed.The transcriptome results indicated that a total of 21177 genes were expressed in both AF and DF and 222 differentially expressed genes(DEGs)were identified.Of them,166 were upregulated and 56 were downregulated genes in DF compare to AF.The function and pathway analysis demonstrated that these DEGs were mainly involved in steroid hormone biosynthesis.Furthermore,a total of 12 differentially expressed mi RNAs were verified between DF and AF.Of them,9 were upregulated and 3 were downregulated.We further selected 4 key candidate mi RNAs(mi R-196,mi R-451-5p,chi-mi R-202-5p and mi R-144-5p)to jointly analyze them with DEGs.The results showed that the key genes were mainly related to steroidogenesis,oocyte meiosis,and metabolic pathway.2.Exocellular vesicles(EVs)were isolated from goat ovarian follicular fluid(FF)and the EVs affecting the follicle cells were investigated.EVs were isolated by using gradient centrifugation and ultra-centrifugation,and the exosomes were identified by WB,Transmission Electron Microscope(TEM),and Nanosight analysis,indicating that exosomes were existing in FF.Di I-labeled exosomes can be captured by granulosa cells(GCs)and cumulus cells of(COCs).Furthermore,EVs isolated from ovarian follicles with different stages [small follicular follicles(SFFs;dimeter<3 mm)and large follicular follicles(LFFs;dimeter>5 mm)],SFF-EVs could improve GCs proliferation and both SFF-EVs and LFF-EVs could induce cumulus expansion after the co-culture with GCs and COCs in vitro.3.To further explore small RNA(s RNA)content in FF exosomes,6 exosomes s RNA libraries were constructed for s RNA sequencing.The results showed that different mi RNA expression profiles between SFF-EVs and LFF-EVs.A total of 285 known mi RNAs were identified in FF exosomes.The top 23 highly expressed mi RNAs were occupied 96% and 97% in SFF-EVs and LFF-EVs,respectively.Among the top 23 mi RNAs in the two groups(SFF-EVs and LFF-EVs),a total of 30 mi RNAs were in account.Among them,14 were differentially expressed mi RNAs,8 were upregulated and 6 were downregulated mi RNAs.Chi-mi R-202-5p was the most significantly differentially expressed mi RNA in LFF-EVs.Furthermore,a total of 1968 target genes were predicted and involved in Axon guidance,Fox O signaling,MAPK signaling,and PI3K-AKT signaling pathway.4.Chi-mi R-202-5p functionally repressed GCs proliferation and induced apoptosis in vitro.Based on the abovementioned results,chi-mi R-202-5p was selected as a candidate mi RNA for further research.The result of quantitative PCR(q PCR)showed that chi-mi R-202-5p was especially highly expressed in GCs and cumulus cells that were isolated from LFFs.FACS analysis and 5-Ethynyl-2’-deoxyuridine(Ed U)assay indicated that the chi-mi R-202-5p overexpression induced GCs apoptosis and suppressed proliferation.In contrast,chi-mi R-202-5p inhibitors decreased the apoptosis of GCs and improved GCs proliferation.These results demonstrated that the chi-mi R-202-5p is a functional mi RNA in GCs.5.To verified the mechanism of chi-mi R-202-5p in GCs,three chi-mi R-202-5p binding sites on the 3’-UTR of transforming growth factor beta receptor 2(TGFβR2)m RNA were predicted.The relative luciferase activity showed that chi-mi R-202-5p targets TGFβR2 m RNA by binding to seed regions.The chi-mi R-202-5p overexpression in GCs suppressed TGFβR2 m RNA and protein levels and further decreased downstream protein(SMAD3)phosphorylation,leading to GCs apoptosis.6.We further identified the reason behind the high expression of chi-mi R-202-5p in GCs in large follicles.The results demonstrated that steroidogenic factor 1(SF1)increased the chi-mi R-202-5p and CYP19A1 m RNA expression by binding to their promoter regions and increased estrogen E2 production in GCs.Furthermore,the chimi R-202-5p-mediated SF1 attenuated the TGFβR2 expression,leading to a negative regulation of TGF-β signaling pathway in GCs.In summary,we used RNA sequencing to explore the relationship between m RNAs and mi RNAs in AF and DF of Shaanbei white cashmere goats and identified the mi RNA profile of FF exosomes from ovarian follicles with different stages.We further investigated chi-mi R-202-5p as a functional mi RNA regulating TGF-β signaling pathway in goat GCs.Collectively,our data provides a foundation for molecular mechanism of goat ovarian follicle development that will increase the understanding of goat reproduction. |
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