Expression And Function Of Bone Morphogenetic Protein 6 In Chicken Follicle

BMP6(Bone Morphogenetic Protein 6)is a member of BMPs protein family,is a secreted multifunctional protein,studies have shown that the expression of BMP6 in mammalian animal ovary and can regulate follicular steroid hormone synthesis,but differences exist in different species.Therefore,on the basis of these findings,we studied the expression and function of BMP6 in chicken follicles.Firstly,using fluorescence quantitative PCR and Western blot analyzed the expression level of BMP6 mRNA in different stages of follicle and chicken protein;in cultured chicken granulosa cells in vitro,protein gradient of different concentration,the proliferation of granulosa cell analysis by CCK-8 method;the expression of key genes were used to detect the changes in progesterone synthesis and StAR,3P-HSD in different concentration of BMP6 protein in the group treated by fluorescence quantitative PCR and Western blot;cultured granulosa cells of chicken egg,add BMP6 and FSH protein with different concentrations,secretion by Elisa determination of progesterone in the supernatant of granulosa cells.From the above series of experiments,we come to the following main results:(1)Analysis of the expression level of BMP6 mRNA in the follicles of chicken in different period by using fluorescence quantitative PCR,BMP6 mRNA in the Prehierarchical follicles and follicular tissues,and with mature expression gradually rising trend,followed by Western blot analysis of the expression level of BMP6 protein in chicken follicles in different period BMP6,found in the follicular on protein expression of BMP6 and mRNA is similar in is expressed in all stages of the follicles,and the expression of F1 and F2 in the follicles was significantly higher than that in other stages of the follicles.These results indicate that the expression of BMP6 is related to the development of follicle,and it is speculated that BMP6 may be involved in the regulation of the development of chicken follicles and the selection of the dominant follicle and ovulation.(2)In the process of cultivation of chicken granulosa cells in vitro,and set up 4 of the protein in different concentration gradient,respectively Ong/ml(control group),25ng/ml,50ng/ml and 100ng/ml,48h were cultured in vitro,analysis of proliferation of granulosa cells by CCK-8 method.Compared with the experimental group added 25ng/ml BMP6 protein with the cointrol group,the number of granule cells had no obvious change,but the experimental group compared with 50ng/ml and 100ng/ml protein with the control group,the number of granule cells increased significantly. suggesting that BMP6 protein can promote the proliferation of chicken granulosa cells in vitro.(3)For the training of BMP6 protein in chicken granulosa cells during in vitro effect on progesterone synthesis,selection of key protein StAR and 3 beta-HSD two progesterone synthesis,using real-time quantitative PCR and Western blot at mRNA and protein level detected whether BMP6 affect the expression of StAR and 3?-HSD gene.The experimental results showed that exogenous BMP6 could significantly promote the expression of chicken granulosa cells and StAR,3?-HSD gene at mRNA and protein level,and with the increase of BMP6 concentration,this effect has been strengthened,suggest that BMP6 expression in a dose-dependent manner and the regulation of StAR 3 beta-HSD gene,StAR and the 3?-HSD are two key proteins of progesterone synthesis process,so we speculate that BMP6 can promote the synthesis of progesterone in chicken granulosa cells.(4)Isolated and cultured granulosa cells of chicken egg,and add the BMP6 protein with different concentrations,the secretion of progesterone,granule cell supernatant by Elisa showed that the secretion of granulosa cell progesterone added BMP6 protein in granulosa cells was significantly higher than that without the addition of BMP6 protein,and the secretion of progesterone with BMP6 increased significantly increased.In summary,BMP6 both at the mRNA level and the protein level,can show the ability to promote the development of follicles,while the proliferation of granulosa cells and progesterone synthesis has a different degree of regulation.