Effects Of RAF-ERK1/2 Pathway On Synthesis Of Steroid Hormone In Ovarian Granulosa Cells

Ovarian granulosa cells are the most important somatic cells in follicles during the process of synthesis of steroid hormones.The granule cells would promote the development of follicles and provide nutrition and various growth factors for the maturation of oocytes.The apoptosis of granulosa cells is one of the most important reasons of follicular atresia.To explore the feature of granular cell can help us investgate the female ovarian function,follicular development and oocyte maturation.The technology of culturing granular cells in vitro has gradually experienced and systematic,which can help people further study the function of granular cells.The RAF-ERK1/2 pathway is widely expressed in eukaryotic cells and is involved in the mediation of gene expression,cell proliferation,cell metabolism,apoptosis,immune responses and many other important cellular life activities.Although the two key factors RAS and RAF mutations have been found in many kinds of human cancers.Studies have also confirmed that proper activation of the RAS/RAF/ERK1/2 pathway is essential for follicular development and ovulation.It has also reported that the activation of ERK1/2 is a common process that promotes the synthesis of steroid hormones.But there is not any related research has not been reported about the function of RAF/ERK1/2 pathway in the bovine ovary.The regulation of steroid hormone synthesis in ovarian granulosa cells by RAF-ERK1/2 pathway is unclear.In this study,we serve bovine ovarian granulosa cells as the experiment material.,and used the RAF specific inhibitors(GSK2118436)and the ERK1/2 specific inhibitor(SCH772984).The two inhibitors were used to inhibit RAF-ERK1/2 pathway in granulosa cells.The experiment mainly used immunohistochemistry,qRT-PCR,ELISA Western-Blotting,and other experimental methods to try to explore the influence of RAF-ERK1/2 pathway of steroid hormones in bovine ovarian granulosa cells.The main result is the following:1.B-raf and C-raf were both expressed in bovine ovary tissues and were highly expressed in granulosa cells.The serum-free culture system for bovine ovary granule cells in vitro was established while the purity of granulosa cells which was separated and cultured in experiments was 96%.2.In vitro granulosa cells were cultured by adding 1 μM,2 μM and 4 μM inhibitors GSK2118436.The qRT-PCR was used to detect the expression of B-raf,C-raf and ERK1/2.The results showed that after the addition of the inhibitor,the expression of B-raf,C-raf,ERK1 and ERK2 were significantly decreased(P<0.05).Western-Blotting results revealed that the inhibitor can also significantly inhibit the expression of B-raf,C-raf and ERK1/2,which is consistent with the mRNA expression.The ELISA results indicated that the inhibitor GSK2118436 significantly promoted the synthesis of progesterone and inhibited the secretion of testosterone and estradiol(P<0.05).Obviously,the 4 μM inhibitor GSK2118436 was the best concentration in this assay.3.During the cultivation of granulosa cells in vitro we adding 0.2 μM,1 μM and 5 μM inhibitor SCH772984.The qRT-PCR showed that the inhibitor significantly up-regulated the expression of B-raf and C-raf with the tendency of negative feedback regulation(P<0.05)while the inhibitor down-regulated the expression of ERK1/2(P<0.05).Western-Blotting results indicated that the inhibitor SCH772984 significantly promoted the expression of B-raf and C-raf and inhibited the expression of ERK1/2.ELISA results revealed that the inhibitor SCH772984 significantly promoted the synthesis of progesterone and inhibited the secretion of testosterone and estradiol(P<0.05).In summary,the most effective inhibitor concentration of SCH772984 is 1 μM.4.The most efficient concentration of the two inhibitors,4 μM GSK2118436 and 1 μM SCH772984 were used separate or together in bovine ovarian granulosa cells.ELISA results showed that the co-treatment significantly promoted the synthesis of progesterone and inhibited the secretion of testosterone and estradiol(P<0.05).The amount of progesterone synthesis in the co-adding group was significantly increased compared with that in the erk1/2 inhibitor group alone(P<0.05).There was no significant difference between the other group(P > 0.05).The qRT-PCR results revealed that the two inhibitors down-regulated the expression of StAR and CYP17 and promoted the expression of CYP11 and HSD3(P<0.05).There was significantly decreased difference of the CYP17 expression after co-addition compared with the other two groups(P<0.05).There was not any significantly difference among any other treatment groups(P>0.05).In summary,B-raf and C-raf are expressed in bovine ovary tissues and mostly expressed in granulosa cells.It showed that the expression of B-raf and C-raf were promoted by the inhibition of ERK1/2 with the negative feedback control.At the same time,the expression of StAR and CYP17 were interrupted while the expression of CYP11 and HSD3 were accelerated by restraining ERK1/2.RAF promotes the synthesis of progesterone and hindered the secretion of estradiol and testosterone through ERK1/2.