The Expression Of FOXO3 Gene In Chicken Follicles And Its Effects On Proliferation And Apoptosis Of Ovarian Granular Cells

FOXO3,also known as forkhead transcription factor 3a,is a member of the O subfamily in the family of forkhead box?FOX?.Studies have shown that FOXO3 gene can be negatively regulated by phosphorylation of PI3k/Akt signaling pathway.The activation of FOXO3 gene is also activated by PI3k/Akt signaling pathway,resulting in cell apoptosis.In mammalian ovaries,FOXO3 gene regulates the atresia and the growth of follicles by promoting apoptosis of ovarian granular cells.For this purpose,we speculate that FOXO3 gene may be an important regulatory factor leading to the apoptosis of avian follicular granular cells.Therefore,this experiment used poultry as the research object.First,we detected the m RNA and protein expression of FOXO3gene in different stages of chicken follicles by using fluorescence quantitative PCR and immunofluorescence technology.Secondly,in the ovary granular cells of the chicken,the m RNA expression of related apoptotic genes in the downstream of PI3K/Akt pathway and three hormone receptor genes were detected after the interference of FOXO3 gene.In addition,different concentrations of FOXO3 protein were added in granular cells of the chicken in order to detect the the m RNA expression of downstream related apoptotic genes and three hormone receptor genes in the downstream of PI3K/Akt pathway.Finally,by adding different concentrations of E2,FSH and LH hormones,we detected the proliferation of chicken ovary granular cells,and the m RNA expression of the three hormone receptor genes.The results showed that:?1?FOXO3 gene expressed in different organizations and all stages of follicles in chicken.The expression quantities of FOXO3 gene in chicken follicles were increased with the increase of follicles.Thereinto,the expression of FOXO3 gene was lowest in F1,significantly lower in the follicle in F2 and F3 level?P<0.05?,and extremely significantly lower in the F4 and F5?P<0.01?.The expression in the F4 and F5follicle was not significant?P>0.05?.The expression of FOXO3 gene in LWF was significantly lower than that of SYF?P<0.01?,and the expression level in SWF was significantly different?P>0.05?.?2?After interfering FOXO3 gene by si RNA in ovarian granular cells,the expression of apoptosis related gene BCL2L11,Bnip3,CDKN1B and FASLG,did not increase or decrease significantly?P>0.05?,and the expression of FSH,LH,E2receptor gene FSHR,ER?,LHR,did not increase or decrease significantly,either?P>0.05?.?3?After adding FOXO3 protein in chicken primary ovarian granular cells,the expression of FOXO3 gene up-regulated significantly.Thereinto,the expression of FOXO3 gene in high concentration treatment group is significantly higher than low concentration group?P<0.05?,while the difference between the low concentration treatment group and the high concentration group was not significant?P>0.05?.The expression level of Bnip3 gene in the high concentration treatment group was significantly higher than that in the other groups?P<0.05?,while the differences in other groups were not significant?P<0.05?.CDKN1B gene was significant different in high concentration treatment groups?P<0.05?.However,the expression of BCL2L11and FASLG genes were no significant differences in different concentrations groups treated with FOXO3 exogenous proteins?P>0.05?.There were no significant differences in expression of the related hormone receptor gene ER,FSHR and LHR?P>0.05?.?4?After adding different concentrations of E₂,FSH and LH in the chicken granular cells,the proliferation of ovarian granular cells occurred in different degrees?P<0.05?.In E₂,FSH-treated granulosa cells,granulosa cells in the high-concentration treatment group significantly proliferated?P<0.01?;in the LH-treated granulosa cells,granulosa cells appeared apoptosis?P<0.05?.Adding different concentrations of E₂resulted in a significant up-regulation of ER?gene expression?P<0.05?,and FOXO3gene expression was significantly downregulated?P<0.05?.The addition of different concentrations of FSH led to a significant increase in FSHR gene expression in the treated group?P<0.05?.The expression of FOXO3 gene in the control group was significantly higher than that in the control group?P<0.05?;LH gene expression was significantly up-regulated after adding different concentrations of LH?P<0.05?,and the expression level of FOXO3 gene in different concentration groups.The difference was not significant?P>0.05?.In summary,FOXO3 gene is expressed in chicken tissues,follicles,and chicken ovary granulosa cells,and it has the function of promoting apoptosis of chicken granulosa cells,but it cannot regulate the receptor gene ER?,FSHR.LHR,therefore the regulation of E₂,FSH,LH and FOXO3 in chicken follicles remains to be further explored.The results of this experiment can provide a reference for a more in-depth exploration of the regulation of FOXO3 gene in chicken follicular development and reveal the molecular mechanism of chicken follicular development differences.