Effect Of AMHR2 On Progesterone Synthesis In Granulosa Cells During Follicular Selection In Chickens

Chicken follicular development has the characteristics of continuous development,priority arrangement and no luteinization after ovulation.Optimal reproductive efficiency and egg production are achieved when follicular development follows a very strict hierarchy of priorities.Follicular selection occurs in the pool of prehierarchical follicles（6–8 mm）and on a daily or near-daily one follicle is selected to become a hierarchical follicle.Granulosa cells from selected follicles began to differentiate,and the sensitivity of granulosa cells to follicle stimulating hormone（FSH）was significantly increased,and the stimulation of FSH caused granulosa cells to start secreting progesterone.In the process of mammalian follicular selection,the sensitivity of granulosa cells to FSH is considered to be inhibited by anti-müllerian hormone（AMH）,which in turn inhibits the development of preantral follicles.However,it is uncertain whether the molecular mechanism and function of AMH in chicken follicular selection are similar to that in mammals.In this research,the intensity of AMH stimulation to granulosa cells was changed by adding exogenous chicken AMH or specific knockout of AMHR2 gene in vitro.The effect of AMHR2 on progesterone synthesis in granulosa cells during follicular selection in chickens was explored,that provide theoretical basis for the regulation of AMHR2-mediated AMH in chicken follicular selection.The results of this research as following:1.To explore the effect of exogenous AMH on chicken follicular granulosa cells,cells from 6–8 mm follicles granulosa layer were cultured with different concentration of AMH（0,5,10,20,40,80 ng/mL）.P4 concentration in culture medium and the related genes expression in granulosa cells were then quantified by ELISA and qPCR.The results showed that 20 ng/mL AMH promoted the expression of P4 synthesis related genes in granulosa cells.AMH plays a key role in follicular FSH threshold.AMH can reduce FSH threshold and promotes FSH stimulation of 6–8 mm follicle at a relatively sensitive concentration,thus affecting the selection of dominant follicles and progesterone synthesis.2.The specific knockout system of chicken AMHR2 gene was successfully constructed,which including a pair of knockout expression vectors of two target sites on the same exon of chicken AMHR2 gene which distance less than 30 bp（pX330-U6-AMHR2_dBsa I-CBh-hSpCas9）,and a corresponding SSA-RPG dual-fluorescent reporting vector（pB-CMV-DsRed-CAG-AMHR2.200bp repeat.Puro-T₂A-GFP）.The knockout efficiency was detected by transfection of HEK293T cells and DF-1 cells.After puromycin enrichment,AMHR2 gene was effectively knocked out at the genome level（⁶0%）.3.Granulosa cells from 6–8 mm follicles were co-transfected by chicken AMHR2 gene knockout expression vector and the corresponding dual-fluorescence reporter vector.The expression of progesterone synthesis related genes in the enriched positive granulosa cells was detected by qPCR,and the results showed that there was a positive correlation between AMHR2 gene and progesterone synthesis related genes.In conclusion,AMHR2 gene is positively correlated with progesterone synthesis related genes in chicken follicular granulosa cells,but the specific molecular mechanism involved in the regulation of progesterone synthesis in granulosa cells still needs further experimental verification.By exploring the effect of AMHR2 on progesterone synthesis in granulosa cells,this study provides a theoretical basis for the regulatory factors and mechanism of chicken follicular selection,and helping to further understand the molecular mechanism of chicken follicular development and selection.