Molecular Mechanism Of CTNNB1 Gene Affecting Proliferation And Sex Hormone Secretion Of Chicken Follicular Granulosa Cells

The laying performance of poultry is affected by many factors,and the development of ovarian follicles is the key factor.Wnt/β-Catenin signaling pathway can regulate ovarian follicle development.β-Catenin protein,which is encoded by CTNNB1 gene,is the core component of this signaling pathway.Our previous research found that the expression levels of CTNNB1 in ovarian tissues from laying/mature samples of same age chickens were higher than that of pre-laying chickens of the same age,and the expression of CTNNB1 gene in granulosa cells of graded follicles was significantly higher than that in membrane cells.This experiment aims to explore the function and molecular mechanism of CTNNB1 gene.The effects of CTNNB1 gene on the proliferation,apoptosis and hormone secretion of chicken grade follicular granulosa cells were investigated by interfering with and overexpressing CTNNB1 gene.The overexpression vectors of transcription factors TCF-4 and SF-1 were constructed and co-transfected with CTNNB1 overexpression vector into chicken grade follicular granulosa cells to study whether CTNNB1 interacts with TCF-4 and SF-1.The results were as follows:（1）The m RNA and protein levels of CTNNB1 gene were significantly increased after transfection of pcDNA3.1-CTNNB1 overexpression vector（P<0.05）.The m RNA and protein levels of CTNNB1 gene were significantly decreased after transfection of si RNA-CTNNB1（P<0.05）.The above results show that pcDNA3.1-CTNNB1overexpression vector and the si RNA-CTNNB1 interference RNA were successfully constructed.（2）The results showed that pcDNA3.1-CTNNB1 could promote the proliferation of graded follicular granulosa cells（P<0.01）,promote the expression of cell proliferation-related genes Cmyc and CCND1（P<0.05）,and promote the cell cycle from G1 phase to S phase（P<0.05）.pcDNA3.1-CTNNB1 could promote the expression of hormone secretion related genes St AR,CYP11A1 and CYP19A1（P<0.05）,and promote the secretion of E₂ and P₄（P<0.05）.pcDNA3.1-CTNNB1 has no effect on the apoptosis of chicken granulosa cells and the expression of apoptosis-related genes Bcl-2 and BIRC5（P>0.05）.When si RNA-CTNNB1 interference RNA was transfected,the result was the opposite.The above results showed that CTNNB1 gene promoted the development of chicken grade follicular granulosa cell cycle from G1 phase to S phase,and then promoted the proliferation of granulosa cells and the secretion of E₂ and P₄,but did not affect the apoptosis of granulosa cells.（3）Co-transfection of CTNNB1 and TCF4 into chicken grade follicular granulosa cells showed that compared with co-transfection of pcDNA3.1 and pcDNA3.1-TCF4 group,co-transfection of pcDNA3.1-CTNNB1 and pcDNA3.1 group and single transfection of pcDNA3.1 group,co-transfection of pcDNA3.1-CTNNB1 and pcDNA3.1-TCF4 group can significantly promote the proliferation of graded follicular granulosa cells（P<0.05）,promote the development of cell cycle from G1 phase to S phase（P<0.05）,and significantly promote the expression of cell proliferation-related genes Cmyc and CCND1（P<0.05）.The results of this experiment showed that CTNNB1 gene and transcription factor TCF-4 have synergistic effect,and jointly affect the proliferation of chicken grade follicular granulosa cells.（4）Co-transfection of CTNNB1 and SF1 into chicken grade follicular granulosa cells showed that compared with co-transfection of pcDNA3.1 and pcDNA3.1-SF1 group,co-transfection of pcDNA3.1-CTNNB1 and pcDNA3.1 group and single transfection of pcDNA3.1 group,co-transfection of pcDNA3.1-CTNNB1 and pcDNA3.1-SF1 group can significantly promote the secretion of E₂ and P₄（P<0.05）and the expression of hormone secretion related genes St AR,CYP11A1 and CYP19A1（P<0.05）.The results of this experiment showed that CTNNB1 gene and transcription factor SF-1 have synergistic effect,and jointly affect the sex hormone secretion of chicken grade follicular granulosa cells.In conclusion,CTNNB1 gene promotes the development of chicken grade follicle granulosa cell cycle from G1 phase to S phase in chicken grade follicles,and then promotes the proliferation of granulosa cells and the secretion of E₂ and P₄,but did not affect the apoptosis of granulosa cells.CTNNB1 gene respectively works with TCF-4 and SF-1transcription factors and affects granulosa cell proliferation and sex hormone secretion.