The Effect Of BMP6 On Gene Expression In Human Granulosa-Lutein Cells Before Or After Ovulation And The Signaling Pathways Underlying

Bone morphogenetic protein 6(BMP6)is a member of the transforming growth factor-b(TGF-b)superfamily that is highly expressed in mammalian oocytes and granulosa cell.This intra-ovarian paracrine factor plays a critical role in regulating the development of ovarian follicles and follicular functions.The expression of BMP6 accumulated in the follicles as follicles growth up.While in the dominant follicle,the expression of BMP6 decreased.After ovulation,the concentration of BMP6 increases gradually.Genetic deletion of BMP6 gene in female mice have a phenotype of reduced ovulation rate,impaired oocyte quality and embryo implantation,leading to decreased litter size.In vitro studies in rats have shown that BMP6 attenuated FSH-induced c AMP activity,production of progesterone and its related steroidogenic enzymes,such as steroidogenic acute regulatory protein(St AR)and P450 side-chain cleavage enzyme.In human granulosa-lutein cells,treatment with BMP6 increased the expression of anti-Müllerian hormone and FSH receptor,whereas it decreased the expression of St AR.In human luteinized steroidogenic cells,BMP6 suppressed luteinization by decreasing the production of progesterone.In human corpus luteum,the expression of BMP6 was increased,which may act as a luteolysis mediator.Taken together,all these studies indicate that BMP6 is an essential intraovarian regulator of follicular functions both in the aspects of maturation of the oocyte and the luteolgenesis.This experiment focused on the role of BMP6 influence on the growth factors closely related to ovulation and corpus luteum formation.1.BMP6 down-regulates GDNF expression in human granulosalutein cellsBone morphogenetic protein 6(BMP6)is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells.Glial cell line-derived neurotrophic factor(GDNF)is an intraovarian neurotrophic factor that plays an essential role in regulating mammalian oocyte maturation.The aim of this study was to investigate the effect of BMP6 on the regulation of GDNF expression and the potential underlying mechanisms.An established immortalized human granulosa cell line(SVOG cells)and primary human granulosa-lutein cells were used as cell models.Our results showed that BMP6 significantly downregulated the expression of GDNF in both SVOG and primary human granulosa-lutein cells.Using dual inhibition approaches(kinas receptor inhibitor and small interfering RNA knockdown),our results showed that both ALK2 and ALK3 are involved in BMP6-induced down-regulation of GDNF.In addition,BMP6 induced the phosphorylation of SMAD1/5/8 and ERK1/2 but not AKT or P-38.Among three downstream mediators,both SMAD1 and SMAD5 are involved in BMP6-induced downregulation of GDNF.Moreover,concomitant knockdown of endogenous SMAD4 and inhibition of ERK1/2 activity completely reversed BMP6-induced down-regulation of GDNF,indicating that both SMAD and ERK1/2 signaling pathways are required for the regulatory effect of BMP6 on GDNF expression.Our findings suggest an additional role for an intrafollicular growth factor in regulating follicular function through their paracrine interactions in human granulosa cells.2.BMP6 increases CD68 expression by up-regulating CTGF expression in human granulosa-lutein cellsBone morphogenetic protein 6(BMP6)is a critical regulator of steroidogenesis that could prevent premature luteinzation before ovulation and regulate the luteolysis after ovulation.CTGF is a multifunctional regulator that plays an important role in regulating mammalian luteolgenesis and follicular function.CD68 is an intraovarian marker of macrophage which could play an important role in physiological regression of the corpus luteum.The aim of this study was to investigate the effect of BMP6 on the expression of CTGF and the subsequent increase in CD68 expression as well as the underlying mechanisms.Primary and immortalized(SVOG)human granulosa cells were used as cell models which were obtained from infertile women undergoing IVF procedure.The expression of CTGF and CD68 were examined after exposure to recombinant human BMP6.Our data showed that BMP6 significantly upregulated the expression of CTGF and CD68 in both SVOG and primary human granulosa-lutein cells.Using BMP I receptor inhibitors(dorsomorphin,DMH-1 and SB431542),we demonstrated that both ALK2 and ALK3 are involved in BMP6-induced up-regulation of CTGF.Additionally,using small interfering RNAs targeting SMAD4 to knockdown of endogenous SMAD4 reversed the BMP6-induced upregulation of CTGF and CD68,indicating that the canonical SMAD signaling pathway is required for the regulatory effect of BMP6 on CTGF and CD68 expression.Moreover,knockdown of CTGF by si CTGF abolished the BMP6-induced up-regulation of CD68 production.The upregulation of CTGF expression mediates BMP6-induced increases in CD68 production in human granulosa-lutein cells through type I receptor(ALK2 and ALK3)and SMAD signaling pathway.3.The up-regulation of Furin expression mediates BMP6-induced increases in TGF-b1 production in human granulosa-lutein cells.Bone morphogenetic protein 6(BMP6)is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells.Transforming growth factor-b1(TGF-b1)is a key intraovarian regulator that plays an essential role in regulating mammalian follicular function and oocyte maturation.Furin belongs to the subtilisinlike proprotein convertase family that controls the activation of diverse functional proteins by cleaving inactive protein precursors in the secretory pathway.The aim of this study was to investigate the effect of BMP6 on the expression of furin and the subsequent increase in TGF-b1 production as well as the underlying mechanisms.Primary and immortalized(SVOG)human granulosa cells obtained from infertile women undergoing IVF procedure were used as cell models.The expression of furin and the production/accumulation of TGF-?1 were examined after exposure to recombinant human BMP6.BMP I receptor inhibitors(dorsomorphin and DMH-1),small interfering RNAs targeting ALK2/3/6,SMAD4 and furin were used to investigate the underlying molecular mechanisms.BMP6 significantly up-regulated the expression of furin and increased the production of TGF-?1 in both SVOG and primary human granulosa-lutein cells.Using dual inhibition approaches(kinase receptor inhibitor and small interfering RNA targeted knockdown),we demonstrated that both ALK2 and ALK3 are involved in BMP6-induced up-regulation of furin.Additionally,knockdown of furin abolished BMP6-induced increases in TGF-b1 production.Moreover,knockdown of endogenous SMAD4 reversed the BMP6-induced up-regulation of furin,indicating that the canonical SMAD signaling pathway is required for the regulatory effect of BMP6 on furin expression and TGF-?1 production.