Effects Of BMP2 On The Growth And Secretion Function Of Porcine Follicular Granulosa Cells

The growth and differentiation of granulosa cells are essential for early follicle initiation and development.Various hormones secreted by granulosa cells can also promote follicle development.Numerous studies have shown that granulosa cell apoptosis is the leading cause of follicular atresia,which leads to a decrease in the number of dominant follicles,leading to premature ovarian failure and causing reproductive disorders in animals.There have been many reports on the regulatory function of BMP2 on granulosa cells,but there are few studies in pigs.This study found that the BMP2 gene was differentially expressed in granulosa cells of porcine follicles treated with ATRA,which also had a regulatory role in follicular development.and it was hypothesized that BMP2 might mediate the function of ATRA on the growth and secretion of porcine follicular granulosa cells.In this study,we used in situ hybridization,seamless cloning,Ed U,MTS,liposome transient transfection,RNA interference,flow cytometry and fluorescence quantitative PCR to investigate the role of BMP2 in the growth and secretion of porcine follicular granulosa cells by detecting changes in the expression of apoptosis,proliferation,cell cycle,granulosa cell differentiation marker molecules and genes related to steroid synthesis.To investigate whether BMP2 mediates the function of ATRA on the growth and secretion of porcine follicular granulosa cells by studying the combined action of RA and BMP2 in porcine follicular granulosa cells.The results of the study were as follows.1.This assay uses a cell culture system that has been developed in the laboratory to isolate porcine follicular granulosa cells and is available for subsequent experiments.In situ hybridization results showed that BMP2 was highly expressed in porcine follicular granulosa cells.After transient transfection of BMP2 interference fragment and overexpression vector for 24 h,the results of fluorescence quantitative PCR showed that interference with BMP2 significantly increased the expression of pro-apoptotic genes such as Caspase3 and BAX,and overexpression of BMP2 significantly inhibited the expression of pro-apoptotic genes such as Caspase3,FASL and BAX.Combined with the flow cytometry results,it was demonstrated that BMP2 inhibited granulosa cell apoptosis;MTS results showed that cell viability was significantly decreased after interference with BMP2 and significantly increased after overexpression of BMP2;Ed U results demonstrated that interference with BMP2 inhibited the proliferation of granulosa cells;fluorescence quantitative PCR results showed that interference with BMP2 significantly decreased the expression of cell cycle-related genes CCND and CDK,and BMP2 overexpression treatment significantly increased the expression of cell cycle-related genes CCND and CDK.Combined with the flow cytometry results,it proved that BMP2 could promote the proliferation process of cells from G1 to S phase.Meanwhile,interference with BMP2 significantly increased the expression of granulosa cell differentiation marker genes LHR and PGR,significantly elevated the expression of steroid synthesis-related genes St AR,CYP11A1,3β-HSD,CYP19A1 and 17β-HSD,and increased the secretion level of progesterone and estrogen;meanwhile,BMP2 overexpression treatment also significantly decreased the granulosa cell differentiation expression of the marker gene LHR,as well as the expression of steroid synthesisrelated genes St AR,CYP11A1,3β-HSD,CYP19A1 and 17β-HSD;significantly reduced the secretion levels of progesterone and estrogen.2.1 nmol/L of ATRA was found to increase the expression of BMP2 m RNA extremely significantly after treatment with a temporal gradient.The expression of apoptosis-related gene BAX in the group transfected with NC fragment followed by ATRA was extremely significantly lower than that in the group transfected with NC fragment followed by DMSO.the MTS results showed that the cell viability in the group transfected with NC fragment followed by ATRA was extremely significantly higher than that in the group transfected with NC fragment followed by DMSO.Fluorescence quantitative PCR results showed that the expression of G1 phase related genes CCND3 and CDK2 was significantly higher in the group transfected with NC fragment followed by ATRA than in the group transfected with NC fragment followed by DMSO.The expression of steroid synthesis-related genes and the secretion level of progesterone were significantly lower in the group transfected with NC fragment followed by ATRA than in the group transfected with NC fragment followed by DMSO,and there was no significant difference in the secretion level of estrogen.On the other hand,the expression of BAX was significantly higher in the ATRA group after BMP2 interference than in the ATRA group after transfection with NC fragments.Fluorescence quantitative PCR results showed that the expression of G1 phase related genes CCND3 and CDK2 was significantly lower in the ATRA group after BMP2 interference than in the ATRA group after transfection with NC fragments.The expression of steroid synthesis-related genes and the secretion levels of progesterone and estrogen were significantly higher in the group with the addition of ATRA after interfering with BMP2 than in the group with the addition of ATRA after transfection with NC fragments.In summary,BMP2 can inhibit the apoptosis and differentiation of porcine granulosa cells,promote the proliferation of granulosa cells from G1 phase to S phase,inhibit the secretion of progesterone and estrogen and the expression level of synthase related genes.Interfering with BMP2 can enhance the role of ATRA in promoting the proliferation of porcine granulosa cells,enhance the role of ATRA in inhibiting granulosa cell apoptosis,and reverse the regulation of progesterone and estrogen secretion and the expression of synthase related genes by ATRA.