Study On The Molecular Mechanism Of FOXL2 In Chicken Granulosa Cell Differentiation

The reproductive performance of hens depends on ovary development,which is a dynamic process controlled by several factors.The mechanism of ovary development and follicle selection in chicken is still unclear.Granulosa cells are the first development cells in follicle,and the differentiation of granulosa cells are associated with follicle selection.Forkhead box L2(FOXL2)is a transcription factor involving in mammalian ovarian development,especially in granulosa cell differentiation.Here,we studied the functions of FOXL2 in the development of chicken ovary.First of all,the expression patterns of FOXL2 in chicken embryo gonad and adult ovary were detected by quantitative PCR(qPCR),whole mount in situ hybridization(WISH)and immunofluorescence(IF).Secondly,the granulosa cells from pre-hierarchical(SGCs)and pre-ovulatory follicles(BGCs)were cultured in vitro,and overexpression of FOXL2 was performed on these cells.To detect the potential transcriptional effect of FOXL2,we used high-throughput sequencing to compare the transcriptomes of SGCs and BGCs overexpressing FOXL2,or not.Lastly,we tested the effect of FOXL2 in the activity of cytochrome P450 aromatase(P450arom/CYP19)promoter by luciferase assay.The main results are as follows:(1)FOXL2 was mainly expressed in female gonad in chicken embryo.In female gonad,the expression of FOXL2 was first detected on E4.5,and increased gradually later.The levels of FOXL2 transcription in male gonads remained low at all of the stages investigated.In adult ovary,FOXL2 transcriptions were expressed throughout all stages of follicle development,especially in granulosa cell layer,but barely expressed in theca layer.The expression of FOXL2 in granulosa cell layer increased in SYF,and kept on a high level in pre-ovulatory follicles(from F5 to F2),then raised dramatically before ovulatory(F1).(2)The expression plasmid of FOXL2 or empty plasmid were transfected in cultured cells.qPCR verified the level of FOXL2 significantly higher in overexpression group compared with control group(more than 20-fold in SGCs and 6-fold in BGCs).(3)RNA-seq analysis revealed that 97,148,313 clean reads and 14,280 genes were presented from eight RNA samples.A pairwise analysis was performed as follows: CTRL-BGCs vs.CTRL-SGCs(Comp.1),FOXL2-SGCs vs.CTRL-SGCs(Comp.2),and FOXL2-BGCs vs.CTRL-BGCs(Comp.3).We found 1,575 differentially expressed genes(DEGs)in Comp.1,including 885 up-regulated and 690 down-regulated when granulosa cells differentiated;207 DEGs in Comp.2(112 FOXL2-activated targets and 95 repressed genes);and 201 DEGs in Comp.3,including 84 induced genes and 117 repressed genes.(4)An analysis of Gene Ontology(GO)term enrichment showed that more than 90% DEGs in Comp.1 related to cellular progress in BP classification,DEGs in Comp.2 related to intracellular signal transduction process,and in Comp.3,DEGs mainly influenced the cell motility,extracellular matrix formation.The KEGG(Kyoto encyclopedia of genes and genomes)pathway analysis showed that main enrichment pathways in Comp.1 were focal adhesion,cell cycle,and ECM-receptor interaction;cytokine-cytokine receptor interaction were main pathway in Comp.2;focal adhesion and ECM-receptor interaction were pathways in Comp.3.(5)To study the major function of FOXL2 in the process of chicken granulosa cell differentiation,1591 genes on both lists of Comp.1 and Comp.2 were selected for analysis.We found that FOXL2 might regulate the differentiation of chicken granulosa cell through focal adhesion and cytokine-cytokine receptor interaction.(6)We found that the expression level of 70 overlapping DEGs in Comp.2 and Comp.3 nearly opposite.To illustrate the effect of FOXL2 in the process of granulosa cell differentiation,41 overlapping DEGs among the three comparisons were selected for analyzing the changes of expression pattern.Two significant gene expression patterns were found using short time-series expression miner(STEM): genes were up-regulated in SGCs either by FOXL2 overexpression or transition into BGCs,but were down-regulated in BGCs following FOXL2 overexpression.(7)The expression pattern of FOXL2 and CYP19A1 were different in chicken ovary follicles.Furthermore,FOXL2 couldn’t regulate the promoter’s activity,but steroidogenic factor 1(SF-1)could active this promoter as a concentration-dependent manner.