Effect Of Chicken FOXL2 On Follicular Granulosa Cells Maintenance And Its Ectopic Expression On Testicular Sertoli Cells

FOXL2 is a member of the fork-head transcription factor superfamily.It is expressed in follicular granulosa cells and plays an important role in regulating ovarian differentiation,development and maintenance.In our previous study,we found that FOXL2 was first expressed in E4.5 days,suggesting that FOXL2 may play an important regulatory role in chicken sex determination and gonadal differentiation.Following follicular development,FOXL2 was low expressed in small white follicles,increased in pre-grade follicular granulosa cells,and increased again in pre-ovulation follicular granulosa cells,suggesting that FOXL2 may play an important role in follicular development.FOXL2 may also play an important regulatory role;therefore,the main purpose of this study is to study the role of FOXL2 in the maintenance of follicular granulosa cells and the effect of ectopic expression of FOXL2 on chicken embryonic testicular Sertoli cells.The results are as follows:1.A pcDNA3.1-FOXL2 overexpression vector with high overexpression efficiency was constructed.After 24 hours of transfection,the relative overexpression efficiency of FOXL2 in DF-1 cells could reach more than 1200 times by quantitative PCR.2.After transfection with pcDNA3.1-FOXL2 vector,the effects of FOXL2 on the proliferation of pre-ovulation granulosa cells(po-GC)were detected by CCK8 and ATP methods.The results showed that the cell viability of pcDNA3.1-FOXL2 group at 48 hours was significantly lower than that of pcDNA3.1 group and blank group,which indicated that over-expression of FOXL2 inhibited the proliferation of pre-ovulation granulosa cells.Meanwhile,48 hours of transfection,the effects of over-expression of FOXL2 on po-GC cycle and apoptosis were detected.The results showed that the proportion of G1 cells in over-expression of FOXL2 group increased significantly,while S and G2 cells decreased significantly and about apoptosis,the proportion of cells in the second and third quadrants increased significantly,which indicated that over-expression of FOXL2 could inhibit the cycle progression of po-GC and promote cell apoptosis.3.To further understand the apoptotic mechanism of overexpression of FOXL2 on po-GC,qPCR detected a large number of key genes related to TNF-a/NF-?B and JNK/MAPK apoptotic pathways.The results showed that over-expression of FOXL2 could significantly up-regulate TNF-a,TNFSF10,FADD,BAK1,DAB21 P,MAP3K7,FAS,MAP3K5,C-JUN,MAPK8,MAPK9,CASP8 and CASP10 expression,which indicated that over-expression of FOXL2 could significantly up-regulate apoptotic genes.Theapoptotic effect on pre-ovulation granulosa cells was mainly through activating JNK/TNF-? signaling pathway.The localization of p65 after overexpression of FOXL2 was detected by immunofluorescence assay.The results showed that p65 was expressed in the nucleus of cultured po-GC regardless of whether FOXL2 was overexpressed or not,which indicated that the apoptotic po-GC could activate the NF-?B cell pathway autonomously.4.Using Target Scan and other software,FOXL2 was predicted to be the target gene of miR-1329-3p.By analyzing the relative quantitative expression patterns of pre-grade granulosa cells(ph-GC)and pre-ovulation granulosa cells,the expression patterns of miR-1329-3p and FOXL2 were found to be opposite,and FOXL2 was initially identified as the target gene of microRNA-1329-3p.Double luciferase reporter gene assay showed that miR-1329-3p could bind to the seed sequence of FOXL2 CDS and significantly inhibit the activity of reporter gene in FOXL2 CDS region.Further analysis by qPCR and Western blot showed that the over-expression of miR-1329-3p could significantly inhibit the expression of FOXL2 gene and protein in transfected granulosa cells,and the results after inhibiting the expression of miR-1329-3p were just opposite to the over-standard.The results showed that miR-1329-3p negatively regulated the endogenous expression of FOXL2 by targeting the seed sequence of its CDS region in granulosa cells.5.Overexpression of FOXL2 in chicken testicular Sertoli cells was detected by radioimmunoassay.The levels of estradiol(E2),progesterone(P)and testosterone in the supernatant of chicken testicular Sertoli cells were detected.It was found that the expression of E2 was significantly up-regulated while the contents of P and T were not significantly changed.Further quantitative PCR was used to detect the expression of genes related to gonad differentiation and development after transfection.The expression of SOX9,DMRT1 and STAT3 genes related to testicular differentiation or development was significantly down-regulated,while CYP19A1,WNT4 and CTNNB1 genes related to ovarian development were significantly up-regulated.Further analysis of the activation of WNT4/beta-catenin signaling pathway in this process was carried out.Overexpression vectors of pcDNA3.1-WNT4 and pcDNA3.1-CTNNB1 were constructed.It was found that,regardless of overexpression of WNT4 or CTNNB1,except DMRT1,SOX9 and STAT3 were significantly down-regulated and CYP19A1 was significantly up-regulated,which was basically consistent with the overexpression of FOXL2 in supporting cells.WNT4/beta-catenin signaling pathway plays a synergistic role;interestingly,theexpression of FOXL2 and CTNNB1 or WNT4 is also significantly up-regulated after overexpression of WNT4 or CTNB1,preliminarily indicating the interaction between FOXL2,WNT4 and beta-catenin.However,whether FOXL2 overexpression plays a role through WNT4/beta-catenin signaling pathway,siRNA interference of beta-catenin showed that the expression of SOX9 increased significantly,FOXL2 and WNT4,CYP19A1 decreased significantly,while the expression of DMRT1 and STAT3 did not change significantly,while knocking down the expression of beta-catenin at the same time of over-expression of FOXL2 showed that the expression of SOX9 in co-transfection group was higher than that in over-expression group of FOXL2 only.The expression of CYP19A1 was significantly up-regulated and CYP19A1 was significantly down-regulated,but the expression of DMRT1 and STAT3 was not significantly different.This indicated that the inhibition of SOX9 by over-expression of FOXL2 was mainly through the WNT4/beta-catenin signaling pathway,and this pathway cooperated with FOXL2 to activate the expression of CYP19A1,but not the main pathway.6.The expression profiles of FOXL2 and CTNNB1 in E4.5-E18.5 gonad of chicken embryo were constructed by qPCR.It was found that the expression of FOXL2 was female-specific and began to express at 4.5 days,and increased gradually with age.The expression of CTNNB1 showed no significant gender trend,suggesting that CTNNNB1 may also paly an important impact on testis development.