The Mechanism Of Amphiregulin On Hormone Synthesis In Human Luteinized Granulosa Cells And Trophoblast Invasion

Amphiregulin(Areg)is an important local regulator,which is expressed in both ovarian granulosa cells and placental trophoblast cells.Among all the EGF family members,Areg shows the highest concentration in both follicular fluid and amniotic fluid in early pregnancy.Previous studies showed that Areg mediated LH/hCG's role on the expansion of cumulus-oocyte complex and oocyte maturation.Meanwhile,Areg could stimulate the synthesis of progesterone in granulosa cells and hCG secretion in syncytiotrophoblasts.Amphiregulin's roles have not been investigated.This study focused on the following three parts to investigate the roles of amphiregulin during luteal phase and early pregnancy:1:To investigate whether Areg regulates the expression of aromatase and the synthesis of E2 in human luteinized granulosa cells and the underlying mechanism;2:To explore whether Areg regulates the expression of VEGF in human granulosa cells;3:To investigate whether Areg regulates the invasiveness of HTR-8/SVneo cells and the mechanism.Part 1 Human chorionic gonadotropin-induced amphiregulin stimulates aromatase expression in human luteinized granulosa cells:a mechanism for estradiol production in the luteal phaseObjective:To investigate whether amphiregulin(Areg)regulates aromatase expression and E2 production in human luteinized granulosa cells.Methods:Primary human luteinized granulosa cells were obtained from women undergoing IVF treatment and used for this investigation.Small interfering RNAs was used to knock down the expression of target factors.Reverse transcription quantitative real-time PCR(RT-qPCR)and western blot were used to measure the mRNA and protein levels,respectively.Follicular fluid,serum samples and culture medium were collected,and ELISA was used to measure the concentration of Areg and E2.Correlation analysis was performed.Results:Treatment of hGL cells with Areg stimulated aromatase expression and E2 production through the activation of AKT pathway.PI3K inhibitor--LY294002 could abolish this upresgulation.Inhibition of EGFR(EGF receptor)activity and EGFR knockdown attenuated hCG-induced up-regulation of aromatase expression and E2 production.Follicular fluid and serum were collected from 65 infertile women during IVF treatment.The protein levels of Areg in the follicular fluid were positively correlated with the E2 levels in serum after 2 days of oocyte pick-up and in the follicular fluid of IVF patients.Conclusion:Areg mediates the hCG-induced up-regulation of aromatase expression and E2 production in human luteinized granulosa cells through the activation of EGFR and AKT signal pathway.Part 2 Amphiregulin could upregulate VEGF expression in human granulosa cellsObjective:To investigate whether Areg regulates the expression of VEGF(Vascular endothelial growth factor)in human granulosa cells.Methods:Human luteinized granulosa cells and cumulus cells were isolated and used for this investigation.RT-qPCR was used to measure the expression level of mRNA.The level of Areg and VEGF levels in follicular fluid,and the levels of VEGF in culture medium were measured using ELISA.Correlation analysis was performed among the predictors of OHSS(Ovarian hyperstimulation syndrome).Results:Treatment of cultured granulosa cells with Areg stimulated VEGF mRNA and secretion.Increased Areg levels were associated with transcript levels and follicular content of VEGF.SiRNA-mediated knockdown of EGFR and AREG attenuated the hCG-induced upregulation of VEGF.hCG could upregulate the expression of Areg and VEGF mRNA more in OHSS granulosa cells than that in control groups.Areg,EGFR,and HER2 transcripts in granulosa cells as well as follicular fluid Areg proteins were elevated in OHSS patients.Follicular fluid Areg protein levels are positively correlated with OHSS risk factors such as basal antral follicle counts(AFC),number of oocytes retrieved,number of MII oocytes,and serum E2 levels on hCG day/oocyte-pick-up(OPU)day/2 days after OPU day.Conclusion:Areg-EGFR/HER2 partially mediates hCG-induced VEGF expression in human granulosa cells.Part 3 Amphiregulin promotes trophoblast invasion by increasing MMP9 activityObjective:To illustrate the role of Areg on trophoblast invasiveness and underlying mechanism.Methods:An immortalized human early extravillous cell line,HTR-8/SVneo,was used for this investigation.Matrigel-transwell invasion assay was used for testing the effects of Areg on cell invasiveness.SiRNA targeting at AREG was used to silence the endogenous expression of AREG.Matrix metalloproteinase-9(MMP9)and Matrix metalloproteinases-2(MMP2)mRNA expression levels and activities were measured using RT-qPCR and zymographic analysis.Cellular signals involved in the invasion process were verified using specific inhibitors and western-blot.Results:Our results showed that Areg could promote HTR-8/SVneo cell invasiveness without interfering cell proliferation,and significantly upregulate the expression of MMP9 and TIMP-1(Tissue inhibitors of matrix metalloproteinases-1)mRNA as well as the ratio of MMP9/TIMP-1 in dose-and time-dependent manners.Using specific inhibitors for MEK and PI3K signalings further indicated that,both ERK1/2 and Akt signal pathways were required for Areg-induced cell invasiveness.The co-ordination between ERK1/2 and Akt signaling pathway was needed for the upregulation of MMP9 mRNA,while ERK1/2 mediate the upregulation of TIMP-1 mRNA.Conclusion:ERK1/2 and Akt signaling pathways mediate Areg-induced upregulation of MMP9 activity,which subsequently contribute to Areg-promoted of HTR-8/SVneo cell invasion.