Mechanism Of Fibroblast Growth Factor 8 And 18 Regulate Bovine Follicular Theca And Granulosa Cells

Fibroblast growth factors(FGFs)play important roles in the development of follicles in a variety of mammals.FGFs regulate pathways through receptor tyrosine kinase(RTK),alter cell proliferation and differentiation.FGFs have been studied on granulosa cells,and the molecular mechanism of regulation of cell signaling pathways,hormone synthesis and apoptosis in theca cells has not been reported.In this study,we used bovine follicular theca cells and granulosa cells,using real-time PCR,radioimmunoassay,enzyme-linked immunosorbent assay,adenovirus vector infection,Western Blot and other techniques to explore the effect of FGF8 and FGF18 on hormone synthesis in two cell types and EGR1's(Early growth response protein 1)function in the FGF response,in order to elucidate the mechanism of FGF8 and FGF18 on the regulation of follicular theca cells and granulosa cells.The main results are as follows:1.Effects of FGF8 and FGF18 on follicular theca cellsAfter 4 days of treatment,10 ng/mL FGF8 significantly reduced the level of apoptotic cells detected by flow cytometry.The concentration of progesterone in the culture medium was determined by radioimmunoassay,and 1 ng/mL of FGF8 inhibited the secretion of progesterone.Enzyme immunoassay was used to analyze the testosterone in the culture medium,and 10 ng/mL FGF8 significantly inhibited the testosterone secretion.Real-time PCR analysis showed that 10 ng/mL FGF8 elevated the mRNA expression of Spry1,Spry2 and Spry4,but had no effect on Spry3.FGF8 up-regulated the mRNA levels of NR4A1 and NR4A3,but did not change NR4A2;FGF8 increased the expression of ETV1,ETV4 and ETV5;FGF8 also up-regulated the expression of EGR1 and EGR3.Western blot analysis showed that MAPK3/1 pathway and AKT pathway were activated within 15 min after FGF8 treatment.10 ng/mL FGF18 significantly down-regulated the apoptotic level of follicular theca cells and inhibited the secretion of progesterone and testosterone.The mRNA levels of Spry1,Spry2 and Spry3 was not affected by FGF18 by Real-time PCR analysis,while Spry4 wasup-regulated but was not significantly different.The expression of NR4A1 and NR4A2 did not change at 8 h after treatment,while the mRNA level of NR4A3 was significantly increased.FGF18 increased the expression of ETV4 at 8 h after treatment.FGF18 also elevated the expression of EGR1 and EGR3.The MAPK3/1 pathway was activated after FGF18 treatment,but the AKT pathway was not activated.2.EGR1 regulated follicular theca cellsEGR1 was overexpressed with adenovirus vector Ad-EGR1 to study the effect of EGR1 on theca cells.Using MOI = 200 virus infected theca cells,the EGR1 protein was successfully increased.Overexpression of EGR1 did not affect MAPK3/1 pathway and AKT pathway.EGR1 up-regulated the mRNA level of Spry1,but did not affect Spry2-4.EGR1 also up-regulated the mRNA levels of NR4A1-3,ETV1,ETV4 and ETV5.EGR1 increased the expression of EGR3.Overexpression of EGR1 down-regulated the expression of CYP11 and CYP17,inhibited the hormone secretion of follicular theca cells and increased the activity of theca cells.The expression of EGR1 gene was down-regulated by siRNA,and the expression level of EGR3,NR4A3 and Spry2 was down-regulated,but the expression of ETV1 did not change.The secretion of progesterone and testosterone did not change after EGR1 silence,but the expression levels of CYP11 and CYP17 were doubled.Interference of EGR1 promoted the apoptosis of theca cell.FGF8 lost the ability to up-regulate Spry2,NR4A3,ETV1 and EGR3 in EGR1 silenced theca cells3.EGR1 regulated granulosa cellsFGF8 and FGF18 inhibited granulosa cells estrogen and progesterone secretion;FGF8enhances activity of granulosa cells while FGF18 inhibits cell's viability.FGF18 had no effect on many FGF response genes,but FGF 18 upregulated the expression of EGR1,indicating that EGR1 may be a key factor in the respone of FGF18.EGR1 protein was successfully increased by Ad-EGR1 infection.Overexpression of EGR1 upregulated the expression levels of Spry1,Spry2 and Spry4,but without effect on Spry3.EGR1 significantly increased the mRNA levels of NR4A1-3,EGR3,FOS and other genes.EGR1 activated the MAPK3/1 pathway by up-regulating EREG and has no effect on the AKT pathway.EGR1 did not affect the apoptosis of granulosa cells,which simultaneously up-regulated the apoptotic genes like P53 and anti-apoptotic gene XIAP and so on.EGR1 inhibited the secretion of estrogen but had no effect on the synthesis of progesterone.In conclusion,FGF8 and FGF18 bind to membrane receptor kinase,activate MAPK3/1or AKT signaling pathway to regulate the expression of transcription factor EGR1 in follicular theca cells,and regulate the expression of related genes,inhibit hormone secretion and regulate theca cell apoptosis.EGR1 plays important roles in FGF response in granulosacells,but the pro-apoptotic effect on granulosa cells of FGF18 is not mediated by EGR1.The results of this study provide theoretical basis for further elucidating the mechanism of FGFs regulate theca and granulosa cells and the regulation mechanism of FGFs during follicular development.