Gene Function Of AMH In Chicken Ovarian Granulosa Cells

As the only known cytokine that inhibit the growth of primordial follicles in female animals,Anti-Müllerian hormone(AMH)can reduce the initial recruitment of primordial follicles by inhibting aromatase activity,and weaken ovarian functionby inhibiting FSH sensitivity of follicle.However,there are few studies on AMH function in poultry.In this study,the expression of AMH gene is changed by knocking out or overexpression in granulosa cells of primary follicles.The expression of relative genes were determinated to analyse the function of AMH in chicken ovarian granulosa cells.1.We constructed the vector of AMH gene knock-out pll3.7-U6-SgAMH-spCas9 and SSA-RPG double fluorescence screening reporter pB-CMV-DsRed-CAG-AMH.200 bp repeat.Puro-T2A-GFP.Co-transfected this two plasmids into chicken DF-1 cell line.The percentage of GFP positive cells in RFP positive cells was 5% without puromycin.T7E1 enzyme digestion results demonstrated that the percentage of mutant cells was significantly increased after puromycin screening,TA cloning sequencing results demonstrated that the mutant efficiency was close to 50% after 3.0 ?g/ml puromycin enrichment.2.The spontaneous apoptosis happened in 3 days if chicken ovarian granulosa cells were isolated and cultured in vitro.Phenotype characterized by FSH treatment effect disappears when granulosa cells were cultured in vitro.The cultured conditions were optimized for hormones,cytokines,vitamins and rat tail collagen.The culture conditions finally used were DMEM media supplemented with 10% FBS,1×NEAA,1×Compound vitamins solution and rat tail collagen as an extracellular matrix,that were capable of supporting GCs passages 4times with their specialty.3.To investigated the role of AMH in Chicken ovarian granulosa cells,we co-transfected AMH knockout vector and SSA-RPG screening reporter into chicken ovarian granulosa cells via Lipofectamine?3000.The transfection efficiency of reporter vector was about 30% and the knockout efficiency was about 5%.The genome was isolated when half of the cells died after 0.5 ?g/ml puromycin enriched.TA cloning sequencing results demonstrated that the mutant rate was about 2%.We constructed overexpression vector pcDNA3.1-AMH.Granulosa cells transfected AMH knockout vector and overexpression vector were further treated with 2.5 units/ml FSH.The gene transcriptional expression dynamics were analyzed after AMH perturbation.The expression of Cyp19a1 gene has changed but Smad5 or ALK2 remain unchanged.This study revealed the function of AMH gene in chicken granulosa cells,which could help us to understand the follicular development and follicle selection in poultry.The results can provide theoretical basis for egg breeding.