The Study Of Bone Morphogenetic Protein 2 In The Regulation Of Ovulation-related Genes Expression And The Underlying Mechanism

The ovulation are complex process tightly controlled by the gonadotropin,involving in oocyte mature,somatic cells growth and differentiation and follicular microenvirment dynamic changes,etc.Besides,the communation of oocyte and surrounding somatic cells also plays a critical role in regulating ovulation.Bone morphogenetic protein 2(BMP2)belongs to the transforming growth factor-?(TGF-?)superfamily.Previous studies have shown that BMP2 is a specific protein derived from ovarian granuosa cells and plays an essential role in the regulation of follicle function.To data,the function of BMP2 during follicular development have been comprehensively studied.But,the roles of BMP2 on ovulation related gene expression in human granulosa cells are still unknow.Here,an established immortalized human granulosa cell line(SOVG)and primary human granulosa-lutein cells were utilized as the research cell model to explore the roles of BMP2 on ovulation related gene expression and RT-qPCR,western blot,ELISA and siRNA,etc were used to examine the underlying molecular and cellular mechanisms and verify the specificity of the effects.The main results were as follows:1.BMP2 inhibits the expression levels of PTX3 in human granulosa cells.The SVOG and primary hGL cells were treated with different concentrations(Ctrl,1,10 and 100 ng/mL)of BMP2 for 12 h.The RT-qPCR and ELISA results showed that BMP2 down-regulated cumulus-oocyte complex(COC)expansion relevant gene PTX3 expression levels in concentration-and time-dependent manner in human granulosa cells.Meanwhile,LH increased PTX3 expression level and BMP2 inhibited basal and LH-induced upregualtion of PTX3 in primary granulosa-lutein cells.Pre-treatment of TGF-? type I receptor inhibitors DMH-1 and dorsomorphin totally abolished BMP2-induced down-regulation of PTX3 expression whereas SB-431542 did not have any effect in SVOG cells.Knockdown of SMAD4 also abolished BMP2-induced down-regulation of PTX3 expression in SVOG cells.2.BMP2 increases mature BDNF production through up-regualtion of BDNF mRNA and Furin expression in human granulosa cells.BMP2 increased BDNF mRNA in both concentration-and time-dependent manner as well as Furin mRNA and protein in SVOG and primary hGL cells.DMH-1 and dorsomorphin pre-treatment also abolished BMP2-induced up-regulation of BDNF mRNA in SVOG cells.Meanwhile,BMP2-induced up-regulation of Furin mRNA and protein expression levels were also inhibited by DMH-1 and dorsomorphin pre-treatment in SVOG cells.Knockdown of SMAD4 also reversed BMP2-induced up-regulation of BDNF mRNA and Furin mRNA and protein expression in SVOG cells.ELISA results showed that BMP2 could increase the production of mature BDNF in human granulosa cells.Knockdown of SMAD4 also inhibited BMP2-induced up-regulation of mature BDNF production.Meanwhile,knockdown of Furin could partially attenuate BMP2-induced up-regulation of mature BDNF production whereas did not affect BMP2-induced up-regulation of BDNF mRNA expression level in SVOG cells.These results indicate that BMP2 could increase mature BDNF production by up-regulating BDNF mRNA expression level and Furin expression levels in human granulosa cells.3.BMP2 induces HAS2 mRNA expression and hyaluronan production through upregulation of CTGF expression in human granulosa cells.In human granulosa cells,BMP2 could increase HAS2 mRNA expression level whereas did not change HAS3 expression level.The production of hyaluronan were also increase after BMP2 treatment.Meanwhile,the mRNA and protein expression levels of CTGF were also upregulated with BMP2 treatment.Knockdown CTGF could totally abolish BMP2-induced upregulation of HAS mRNA expression level and hyaluronan production in SVOG cells,which explainsthat CTGF mediates BMP2-induced up-regulation of HAS mRNA expression level and hyaluronan production in human granulosa cells.4.BMP2 inhibits TGF-?1-induced up-regulation of SMAD2/3 phosphorylation and MMP2 mRNA expression via up-regulation of BAMBI expression.The SVOG cells were pre-treated with BMP2 for 12 h and then additionally treated with TGF-?1 for 30 min,the phosphorylation of SAMD2/3 were examined by western blot.The results showed that BMP2 pre-treatment could inhibit TGF-?1-induced phosphorylation of SMAD2/3.Meanwhile,the SVOG cells were treated with BMP2 and then detect MMP2 expression level at different time points after BMP2 treatment.RT-qPCR results showed that BMP2 can not affect MMP2 mRNA expression level at any time points(3,6 and 12 h)after BMP2 treatment.However,BMP2 pre-treatment inhibited BMP2-induced up-regulation of MMP2 mRNA expression level.Then,SVOG and hGL cells were treated with different concentrations of BMP2 for 12 h and results showed that BMP2 increased BAMBI expression in concentration dependent manner in human granulosa cells.Furthermore,BAMBI mRNA expression level were up-regulated by BMP2 treatment at 3,6,12 and 24 h in human granulosa cells.Knockdown BAMBI could abolish the inhibitory effect of BMP2 on TGF-?1-induced phosphorylation of SMAD2/3 and up-regulation of MMP2.5.ALK2/ALK3-BMPR2/ACVR2 A receptors mediate the function of BMP2 in human granlosa cells.The SVOG and pimary hGL were treated with BMP2 and the phosphorylation of SMAD1/5/8 were detected by western blot.The results showed that BMP2 could increase the phosphorylation level of SMAD1/5/8 at both 30 and 60 min time points.Pre-treatment of DMH-1,dorsomorphin and SB-431542 in SVOG and hGL cells and the additional BMP2 treatment for 60 min.Western blot results showed that DMH-1 and dorsomorphin,but not SB-431542,could totally abolish BMP2-induced phosphorylation of SMAD1/5/8.To further confirm which TGF-? type I are involved in BMP2-induced phosphorylation of SMAD1/5/8,a siRNA-based inhibition approach was used to specifically knockdown the type I receptors,ALK2,ALK3 or ALK6 expression level,separately.Our results show that transfection of the cells with siRNA targeting ALK2,ALK3 or ALK6 for 48 h significantly decreased the mRNA levels of the targeted ALKs.Meanwhile,knockdown of either ALK2 or ALK3 alone significantly attenuated BMP2-induced phosphorylation of SMAD1/5/8,whereas knockdown of ALK6 had no such effect.Notably,the combined knockdown of ALK2 and ALK3 completely abolished the phosphorylation of SMAD1/5/8 induced by BMP2.Knockdown of either ALK2 or ALK3 partially reversed the suppressive effect of BMP2 on PTX3 mRNA levels.However,knockdown of ALK6 did not have this effect.Notably,concomitant knockdown of ALK2 and ALK3 completely reversed the BMP2-induced down-regulation of PTX3 expression.These results indicate that both ALK2 and ALK3 type I receptors are required for the BMP2-induced down-regulation of PTX3 expression.To determine which type II receptor(s)are responsible for the BMP2-induced phosphorylation of SMAD1/5/8,we used specific siRNAs to knockdown endogenous BMPR2,ACVR2 A and/or ACVR2 B.Western blot results showed that the solely knockdown of any of the type II receptors(BMPR2,ACVR2 A an ACVR2B)did not affect the BMP2-induced phosphorylation of SMAD1/5/8.Only the combined knockdown of BMPR2 and ACVR2 A entirely abolished the phosphorylation of SMAD1/5/8 in BMP2-stimulated cells.Besides,knockdown of BMPR2 partially reversed the BMP2-induced down-regulation of PTX3 mRNA.However,there was no such effect when the cells were transfected with ACVR2 A or ACVR2 B.Notably,concomitant knockdown of BMPR2 and ACVR2 A completely reversed the BMP2-induced down-regulation of PTX3 mRNA.Either concomitant knockdown of BMPR2 and ACVR2 B or concomitant knockdown of ACVR2 A and ACVR2 B did not have such effect.These data indicate that both BMPR2 and ACVR2 A type II receptors are required for BMP2 signaling in human granulosa cells.6.SMAD1/5-SMAD4 signal pathway mediates the function of BMP2 in human granlosa cells.The SVOG cells were transfected with 25 nM siSMAD1,siSMAD5 or siSMAD8 for 48 h specifically decreased the targeted SMAD mRNA levels by up to 80–90%.Notably,knockdown of either SMAD1 or SMAD5 alone partially attenuated the stimulatory effect of BMP2 on BAMBI mRNA levels,whereas knockdown of SMAD8 had no such effect.Interestingly,although knockdown of SMAD8 could not affect the BMP2-induced increase in BAMBI mRNA,it increased the basal level of BAMBI mRNA in SVOG cells.Importantly,combined knockdown of SMAD1 and SMAD5 completely abolished the stimulatory effect of BMP2 on BAMBI mRNA expression(Fig.4E).These results indicate that both SMAD1 and SMAD5 are required for the BMP2-induced increase in BAMBI mRNA.To further confirm the role of SMAD signaling in the SVOG cells,we used siRNA to knockdown SMAD4,the common mediator of SMAD signaling.Western blot and RT-qPCR analyses showed that treatment with SMAD4 siRNA decreased the SMAD4 protein and mRNA levels by up to 80%.Importantly,the knockdown of SMAD4 completely abolished the BMP2-induced increase in BAMBI mRNA in human granulosa cells.In summary,BMP2 may prevent LH-induced premature ovulation and luteinization through down-regulating of PTX3 expression in growing follicles.In the oocyte maturation,BMP2 can induce the production of hyaluronan to regulate COC expansion.Meanwhile,BMP2 can also increase BDNF production in granulosa cells to guarantee the normal oocyte maturation.Furthermore,BMP2 regulates the activation of TGF-? signaling pathway and mediates the relevantphysiological effect induced by TGF-?.Our results shed light on thephysiological roles of BMP2 in regulatingfollicular function duringthe periovulatory stage.