Transcriptome Analysis Of MRNA And MiRNA Reveal The Regulatory Mechanisms Of Geese Follicle Selection

In poultry,ovarian follicle selection is defined as a single follicle into the preovulatory hierarchy from a cohort of prehierarchal small yellow follicles,which is tightly associated with egg production performance.The granulosa and theca layers are the principle functional units of ovarian follicles,and their secreted cytokines participate in the regulation of follicle selection in an autocrine/paracrine fashion.However,the roles of the granulosa and theca layers during follicle selection as well as the underlying mechanisms still remain unclear in poultry.Therefore,it is of great theoretic and practical importance to clarify the mechanisms controlling avian follicle selection.The present study investigated the mRNA and miRNA transcriptome profiling in both granulosa and theca layers from geese follicles prior to selection(large white follicles of 4-6 mm in diameter;and small yellow follicles of 8-10 mm in diameter)and subsequent to selection(F5)by transcriptomic sequencing.Through a series of bioinformatics analyses,the key pathways as well as candidate differentially expressed(DE)mRNAs and miRNAs involved in geese follicle selection were identified,and the miRNA-mRNA interaction networks were also visualized.These results finally contribute to revealing the regulatory mechanisms controlling geese follicle selection.The main results obtained in this study were listed as follows:1.Through PCA,the granulosa and theca layers were distinctly separated by the first eigenvector based on the expression profiles of either the genome-wide mRNAs or miRNAs.Compared to the theca layer,three biological samples for the granulosa layer at the same developmental stage were clustered more tightly.Moreover,the granulosa layer showed a wider range of Pearson's correlation coefficient for both mRNAs and miRNAs compared to the theca layer.These results implied that there could be apparent functional differences between the granulosa and theca layers and the granulosa layers may play a more important role than the theca layer during follicle selection.2.For the granulosa layer,a total of 2,782 DE mRNAs were identified during follicle selection(i.e.GHF5 vs.GP8),which was approximately twice the number of DE mRNAs(N=1,524)identified prior to follicle selection(i.e.GP8 vs.GP4).Functional enrichment analysis showed that these DE mRNAs during follicular selection were mainly enriched in the biological processes of "ECM-receptor interaction","tight junction","adherens junction","cell adhesion molecules(CAMs)","focal adhesion","fat digestion and absorption","glycerolipid metabolism","ovarian steroidogenesis",and“steroid biosynthesis".The co-expression network of DE mRNAs during follicle selection was also constructed using the Cytoscape software,showing that 53,15,and 13 genes were related to junctional adhesion,lipid regulation,and steroidogenesis,respectively.In addition,downregulated levels of genes related to junctional adhension may contribute to the reduced molecular barrier between granulosa cells,which enables the entry of keymolecules including vitellogenin into the oocyte to facilitate follicle selection.The dynamic expression patterns of steroidogenesis-related genes may be closely related to the differentiation of granulosa cells before or after follicle selection.3.For the theca layer,415 DE mRNAs were found during follicle selection(i.e.THF5 vs.TP8),which significantly exceeded the number of DE mRNAs identified before follicle selection(i.e.TP8 vs.TP4,N=20).In the theca layer,expression levels ofAGTR1,ANGPTL5,NDNF,SRPX2,ACTG2,THBS1,LPAR3,AGTR2,and FGF13 associated with angiogenesis significantly changed during follicle selection.These changes may be important for theca cell angiogenesis that facilitates the transport of yolk-constituting molecules into the oocytes.4.A total of 117 DE miRNAs were identified in the granulosa layer between GHF5 vs.GP8,which was approximately twice the number of DE miRNAs identified between GP8 vs.GP4(N=60).According to the expression profiles of DE miRNAs during follicle selection,gga-miR-181a-3p,acy-miR-455,etc.,were significantly up-regulated in GP8 vs.GP4,acy-miR-199,acy-miR-2970,acy-miR-191,acy-miR-138,and acy-miR-222,etc.,were significantly changed in GHF5 vs.GP8,whereas gga-miR-29a-3p,gga-miR-124a-3p,and gga-miR-383-5p,etc.,were significantly down-regulated before and after follicle selection(GP4-GP8-GHF5).The dynamic expression patterns of these miRNAs during follicle selection may be closely related to the proliferation and differentiation and steroidogenesis of granulosa cells.5.Fifty-four DE miRNAs were identified in the theca layer between THF5 vs.TP8,which was significantly higher than the number of DE miRNAs identified between TP8 vs.TP4(N=8).In the theca layer,acy-miR-205 and gga-miR-205a were significantly up-regulated in TP8 vs.TP4 group,gga-miR-451,acy-miR-144 and acy-miR-1677 were significantly up-regulated in THF5 vs.TP8 group,gga-miR-10b-5p,gga-miR-135a-5p,and gga-miR-211 were significantly down-regulated in prior to and after follicle selection(TP4-TP8-THF5).The dynamic expression patterns of these DE miRNAs may be related to functional changes in the theca layer during follicle selection.6.The core miRNA-mRNA interaction networks were further visualized in both the granulosa and theca layers.In the granulosa layer,acy-miR-2954,acy-miR-218,acy-miR-2970,acy-miR-100,acy-miR-1329,acy-miR-199,acy-miR-425,acy-miR-181,and acy-miR-147 may be involved in geese follicle selection by regulating the expression levels of junctional adhesion-related target genes,while acy-miR-13 8,acy-miR-107,acy-miR-130,acy-miR-128,and acy-miR-101 may be involved in geese follicle selection by regulating the expression levels of target genes related to lipid metabolism.In the theca layer,gga-miR-15c-5p may be involved in geese follicle selection by regulating expression of angiogenesis-related genes including AGTR2.In conclusion,the process of geese follicle selection is proposed to be simultaneously regulated by DE mRNAs related to junctional adhesion,steroidogenesis,and lipid metabolism in the granulosa layer and those related to angiogenesis in the theca layer.