The Role Of EGF-like Growth Factors And TGF-β1 In The Regulation Of Ovulation-related Genes Expression In Human Granulosa Cells And The Underlying Molecular Mechanism

Part I ContextAberrant regulation of ovulation is one of the major causes of infertility. In animal models, three epidermal growth factor(EGF)-like growth factors, amphiregulin(AREG), betacellulin(BTC) and epiregulin(EREG), have been shown to be involved in ovulation by regulating cyclooxygenase-2(COX-2) expression and prostaglandin E2(PGE2) production. However, whether the same is true in humans remains largely unknown.ObjectiveTo investigate the effects of AREG, BTC and EREG on COX-2 expression and PEG2 production in human granulosa cells. DesignSVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization(IVF) and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effect of AREG, BTC and EREG on ovulation-related functions. Levels of m RNA and protein were examined by RTq PCR and western blotting, respectively. The protein levels of PGE2 were measured by enzyme-linked immunosorbent assay(ELISA). ResultsLH treatment up-regulated AREG, BTC and EREG and COX-2. Knockdown of EGFR attenuated LH-induced COX-2 expression and PGE2 production. Treatment with AREG, BTC and EREG up-regulated COX-2 expression and PGE2 production. The stimulatory effects of AREG, BTC and EREG on COX-2 expression and PGE2 production were blocked by inhibition of EGFR activity and expression. AREG-, BTC- and EREG-activated ERK1/2 signaling, but not Akt signaling, was required for AREG-, BTC- and EREG-induced COX-2 expression and PGE2 production. ConclusionAREG, BTC and EREG induced PGE2 production by up-regulating COX-2 expression through ERK1/2 signaling in human granulosa cells.Part II ContextCyclooxygenase-2(COX-2) expression and prostaglandin E2(PGE2) production Objective have been shown to play key roles in the regulation of ovulation. The transforming growth factor-beta(TGF-β) superfamily members are important molecules that regulate many ovarian functions under normal physiological and pathological conditions. TGF-β1 and its receptors are expressed in human granulosa cells. However, to date, whether TGF-β1 can regulate COX-2 expression and PGE2 production, which in turn contribute to the process of ovulation, remains unknown. ObjectiveTo investigate the effects of TGF-β1 on COX-2 expression and PGE2 production in human granulosa cells. DesignSVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization(IVF) and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effect of TGF-β1 on COX-2 expression and PGE2 production. m RNA and protein levels were examined by RT-q PCR and western blotting, respectively. The concentrations of PGE2 in the culture medium were measured by enzyme-linked immunosorbent assay(ELISA). ResultsTGF-β1 treatment induced COX-2 expression and PGE2 production. The inductive effects of TGF-β1 on COX-2 and PGE2 were abolished by inhibition of TGF-β type I receptor(TβRI). In addition, treatment with TGF-β1 activated Smad2 and Smad3 signaling pathways. Inhibition of Smad signaling pathways by si RNAmediated approaches attenuated TGF-β1-induced COX-2 expression and PGE2 production.ConclusionTGF-β1 induced PGE2 production by inducing COX-2 expression through a Smad-dependent signaling pathway in human granulosa cells.Part III ContextRegulation of progesterone production in granulosa cells is important for normal reproductive functions. Steroidogenic acute regulatory protein(St AR) is recognized as the key regulatory protein involved in the rate-limiting step of steroidogenesis. Transforming growth factor-beta 1(TGF-b1) protein is detected in human follicular fluid, and TGF-b1 and its receptors are expressed in human granulosa cells. However, the functional role of TGF-b1 in the regulation of St AR expression and progesterone production in human granulosa cells remains unknown. ObjectiveTo investigate the effects of TGF-β1 on St AR expression and progesterone production in human granulosa cells. DesignSVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization(IVF) and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effects of TGF-b1 on St AR expression and progesterone production. Levels of m RNA and protein were examined by RT-q PCR and western blotting, respectively. The accumulation levels of progesterone were measured by enzyme-linked immunosorbent assay(ELISA). ResultsTGF-β1 treatment down-regulated St AR expression and decreased progesterone production. The suppressive effects of TGF-β1 on St AR expression and progesterone production were abolished by the inhibition of TGF-β type I receptor(TβRI). In addition, treatment with TGF-β1 activated the Smad2/3 and ERK1/2 signaling pathways. The inhibition of the Smad2/3 and ERK1/2 signaling pathways attenuated the TGF-β1-induced down-regulation of St AR expression and progesterone production. ConclusionTGF-β1 down-regulated St AR expression and decreased progesterone production by activating the Smad2/3 and ERK1/2 signaling pathways in human granulosa cells. Results...