Effects Of BMPR2/SMAD5 Signal Pathway On Proliferation,apoptosis And Steroid Hormone Synthesis Of Chicken Granulosa Cells

Poultry ovulation depends on the normal development of follicles,and the prerequisite for achieving high egg production rate is for follicles to maintain development and continuously mature and ovulate after starting production.The proliferation and apoptosis of follicular granulosa cells play a crucial role in the entire follicular development process,especially in the follicular selection process.They are mainly regulated by endocrine hormones,growth factors,temporal expression genes,and numerous signal transduction pathways.In this study,we sequenced the transcriptome of the normal developing follicles of laying hens and the atretic follicles of nesting hens,and screened the signal pathways that may be involved in regulating chicken follicular development,The addition of exogenous signaling pathway inhibitors affects the growth and development of granulosa cells,in order to clarify the role of signaling pathways in the development of chicken follicular granulosa cells.The main research results are as follows:(1)After transcriptome sequencing of normal follicles of laying hens and atresia follicles of nesting hens,200 genes with significant expression differences were found,including 131 up-regulated genes and 69 down regulated genes,including SMAD5 and BMPR2.GO enrichment analysis shows that it is closely related to cell activation regulation,lymphocyte activation regulation,negative regulation of leukocyte activation,and proliferation in biological processes;The main cell components are intermediate filament cytoskeleton,immune synapse,etc;The main molecular functions are binding and catalytic activity.The KEGG enrichment analysis pathway revealed that the top 20 pathways enriched by differential genes were mainly TGF-β Signal pathways,cell adhesion molecules(CAMs),etc.,among which TGF-β Pathways are closely related to the growth and development of follicles.(2)After interfering with BMPR2 expression in chicken primary granulosa cells cultured in vitro,the m RNA expression levels of cell proliferation related genes PCNA,CDK2,and CCND1 were significantly increased;The CCK8 assay kit showed a significant increase in the proliferation efficiency of granulosa cells;Ed U staining showed a significant increase in the number of positive cells;The expression level of apoptosis related factors(Caspase-3 and Caspase-9)in cells was significantly reduced;Promote the secretion of estrogen(E2)and progesterone(P4)in cells,as well as the expression of related genes CYP11A1 and CYP19A1.The above results indicate that BMPR2 can inhibit the proliferation of chicken granulosa cells and promote cell apoptosis.(3)Overexpression of SMAD5 in chicken primary granulosa cells cultured in vitro resulted in a significant decrease in m RNA and protein expression levels of cell proliferation genes PCNA,CDK2,and CCND1,while the expression levels of apoptosis related genes Caspase-3 and Caspase-9 increased significantly;CCK8 and Ed U detection showed a significant decrease in cell proliferation efficiency,as well as a significant decrease in the synthesis of E4 and P2,as well as the m RNA and protein expression levels of their related genes.However,after interfering with SMAD5 expression in cells,the opposite results were obtained.The above results indicate that SMAD5 can inhibit the proliferation of chicken follicular granulosa cells and promote cell apoptosis.(4)After interfering with BMPR2 expression in primary granulosa cells in vitro,it was found that the expression level of SMAD5 decreased,which can inhibit the BMP/SMAD signaling pathway.After exogenous addition of BMP/SMAD signaling pathway inhibitor LDN193189,it was found that it can promote the proliferation of chicken granulosa cells and inhibit apoptosis by detecting the expression of cell proliferation and apoptosis genes,as well as CCK8 and Ed U detection of cell proliferation efficiency;In addition,it significantly promoted the synthesis of E2 and P4,as well as the gene expression levels of key genes STAR,CYP11A1,and CYP19A1.In summary,the results of this study found that the BMP/SMAD signaling pathway is involved in the proliferation,apoptosis,and steroid hormone synthesis of chicken follicular granulosa cells,providing a theoretical basis for studying the molecular regulatory mechanisms of follicular development in poultry.