Study Of Dehydroepiandrosterone On The Secretion And Its Mechanism Of Poor Ovarian Response Human Ovarian Luteinizing Granulosa Cells

Objective: 1)The aim of the study is to investigate the culturing of the original Poor Ovarian Response(POR)human ovarian luteinizing granulosa cells in vitro and the effect of different concentrations of Dehydroepiandrosterone(DHEA)on its hormone secretion.2)To investigate possible mechanisms of the impact of DHEA on granulosa cells’ oxidative stress,secretion function by treating KGN cell line with different concentrations of DHEA.Methods:1.The original Poor Ovarian Response human ovarian luteinizing granulosa cells were cultured in vitro and the identity was confirmed with FSHR expression: The follicular fluid was collected from the Poor Ovarian Response infertility patients who were treated with in vitro fertilization-embryo transfer(IVF-ET)or intracytoplasmic sperm injection(ICSI)at the Reproductive Medical Center of the Second Affiliated Hospital of Chengdu University of Traditional Chinese Medicine(between October 2015 and January 2016).The granulosa cells from the follicular fluid collected during oocyte retrieval was separated with 40% Percoll separating medium,dissociated with Red Blood Cell Lysis Buffer,washed with PBS to purify and frozen.The frozen granulosa cells were cultured with complete medium with DMEM/F12+15%FBS+1%mycillin.Granulosa cells were identified through cell immunofluorescence staining with FSHR.2.To study the secretion of cultured POR human ovarian luteinizing granulosa cells in vitro with DHEA: the POR human ovarian luteinizing granulosa cells were divided into five groups after being cultured for 48 hours in vitro,and were treated with complete medium with different concentrations of DHEA: negative control(DHEA0um),DHEA 0.05 um,DHEA 0.5um,DHEA 5um and DHEA 10 um.After 24 hours the secretion of E2 were measured by chemiluminescence immunoassay and the protein expression of CYP19A1 gene were measured by Western blot.3.To study the effect of DHEA on secretion function and oxidative stress of KGN cell line :1)To establish an Oxidative stress cell model: the KGN cell line was divided into four groups after being cultured for 48 hours,treated with complete medium with different concentration of H2O2: negative control(1/1000 DMSO,0 um H2O2),50 um H2O2,100 um H2O2 and 200 um H2O2,.The activity of KGN cell line were measured with MTT.The protein expression of BCL-2 and BAX gene were detected by Western blot and the m RNA expression of BCL-2 and BAX gene was determined by QPCR after being treated with the lowest dose dettermined from MTT.2)To treat KGN cell line with DHEA: the ability of scavenging free radicals of DHEA was measured by the means of DPPH;in different oxidative stress state: 0um H2O2,50 um H2O2 and 100 um H2O2.The KGN cell line was cultured for 48 hours and was divided into groups with different concentration of DHEA: negative control(DHEA0um),DHEA0.05 um,DHEA0.5um,DHEA5 um and DHEA10 um.24 hours later the activity of SOD were detected by T-SOD kit.The secretion of E2 were measured by chemiluminescence immunoassay.The protein expression of CYP19A1 gene were examined by Western blot and the m RNA expression of CYP19A1 gene were examined by QPCR.Results:1.The POR human ovarian luteinizing granulosa cells cultured in vitro are similar to the normal human ovarian luteinizing granulosa cells.The cells grow in a single-layer adherent,extending pseudopodia to fusiform after 24 hours.The cells are fully formed and rich in particulate substance after 48 hours to reach a peak of cell proliferation.However,as a whole,the POR human ovarian luteinizing granulosa cells grow slower and the culturing of them is more difficult than normal.2.The POR human ovarian luteinizing granulosa cells cultured in vitro are treated with different concentrations of DHEA for 24 hours.The secretion of E2 in treatment groups are increased compared with the negative control group.The difference is statistically significant except the group of DHEA 0.05um(P<0.05),but the protein expression of CYP19A1 gene of DHEA0.05 um group is significantly higher than that of the negative control group(P<0.05).3.To study secretion function and oxidative stress on KGN cell line with DHEA:1)The secretion of E2 in treatment groups is increased compared with that of the negative control group,and the difference is statistically significant(P<0.05).2)The oxidative stress cell model is established: different concentrations of H2O2 can reduce the growth activity of KGN cell line compared with the group of 0um H2O2.The concentration of 100 um H2O2 and 200 um H2O2 can significantly inhibit the KGN cell line growth(P<0.05),and the lowest dose effective intervention of H2O2 concentration is 100 um.With the increase of concentration of H2O2,the protein expression of BCI-2 tends to reduce whereas BAX tends to rise.The expression of m RNA has no obvious change.3)To treat KGN cell line with DHEA:i There is a weak antioxidant effect of DHEA from DPPH.Regardless of whether KGN cell line is in oxidative stress state,the SOD activity in DHEA0.05 group is higher than that of the negative control group,and the difference is statistically significant(P<0.05).ii The protein and m RNA expression of CYP19A1 gene are obviously higher than that of the negative control group,and the difference is statistically significant(P<0.05).iii Treating KGN cell line at the concentration of 100 um H2O2 using different concentration of DHEA and we can see the trend of down-regulation in all treatment groups with the protein expression of CYP19A1 gene,but the falling range in the group of DHEA0.05 um is lower and the difference is not statistically significant(P>0.05);the m RNA expression of CYP19A1 gene in all intervention groups show no obvious change.Conclusion:1.The POR human ovarian luteinizing granulosa cells,through separation and purification of this experiment,is higher in purity,which proves the feasibility of the experiment scheme and provides a reference for subsequent POR luteinizing granulosa cells culturing in vitro.2.In our study,the secretion of E2 in treatment groups is on the rise after DHEA is added to the POR granulosa cells.Low concentrations of DHEA may not consume too much CYP19A1 activity,making it easier for CYP19A1 protein expression to change;high concentrations of DHEA may synthesize more E2.The CYP19A1 activity may decrease the concentration of substrate increases;however,when the substrate reaches a certain concentration,the secretion of E2 will no longer increase.3.KGN cell line has similar secretory activity to POR human ovarian luteinizing granulosa cells.Oxidative stress status can affect KGN cell line activity to increase its apoptosis,and the apoptosis may be mediated by ERK apoptotic pathways.The antioxidant effect of DHEA is likely weaker.After treating KGN cell line,low concentration of DHEA may show weaker antioxidant activity through the increase of protein expression for CYP19A1 gene.The antioxidant activity and the level of SOD are consistent.The m RNA expression of CYP19A1 and ovarian reactivity may be related.Low concentration of DHEA exhibits the function of antioxidant stress,possibly though increasing the activity of CYP19A1 or increasing its expression when DHEA is converted into E2.4.Our study confirm the secretory function of E2 with DHEA to granulosa cells from patients with POR.It is believed that the underlying mechanism of DHEA improving the ovarian function of patients with POR may be related to antioxidant effect and the increases of substrate concentration,providing an experimental support for using DHEA on clinical patients.