Effects Of GABA On E₂ And P Secretions And Apoptosis By Human Ovarian Granulosa Cells In Vitro

The distribution of L-glutamate decarboxylase(GAD),a major biosynthetic enzyme for gamma-aminobutyric acid(GABA),is well-recognized function as a cerebral.Recently,people have discovered more than thirty kinds of peripheral tissues have GABA,and fallopian tube and ovary have higher GABA concentration than the CNS which indicated a physiological sibnificance of this amino acid in the female reproductive systems.Further study found that in rat ovary have GABA neurons,ovarian tissue has the specificity of GABA binding sites.Animal experiments prompted exogenous GABA on luteinized granulosa cell secretion of estradiol and progesterone have inhibitory effects,and this inhibition may be related to GABA receptor and adenylate cyclase system.The data suggested that GABA can promote cultured rat luteal spontaneous apoptosis of granulosa cells in vitro. Many scholars at home and abroad reported that apoptosis of granulosa cells increasing affected the IVF-ET cycles pregnancy outcomes.We used electrochemiluminescence technique to explore the possible mechanism of exogenous GABA effect on human luteinized granulosa cells secretion of estradiol and progesterone,and used TUNNEL technique to detect how exogenous GABA affect human luteinized granulosa cell apoptosis,and used ELISA technique to detect GABA concentration,to explore the impact of GABA on pregnancy outcomes.Part one Effects of GABA on E₂ and P Secretion by Human Ovarian Granulosa Cells in VitroRecent work suggests that exogenous GABA on rat luteinized granulosa cells secretion of estradiol and progesterone have inhibitory effects,and this inhibition may be related to GABA receptor and adenylate cyclase system.But exogenous GABA whether have the same inhibitory effects on human luteinized granulosa cells secretion of estradiol and progesterone,no one has reported.ObjectiveWe used electrochemiluminescence technique to explore the possible mechanism of exogenous GABA effect on human luteinized granulosa cells secretion of estradiol and progesterone.Methods1.Thirty-two barren patients were treated by IVF/ICSI-ET in southern hospital' s reproductive center.They were less than thirt-five years old.Barren cause was fallopian tube factor or male factor.They were all at their first IVF/ICSI-ET cycle using down regulation long controlled hyperstimulation protocol. Centrifuging and separating luteinized granulosa cells,the isolated luteinized granulosa cells were divided into four groups to carry out in vitro for twenty-four hours,Discarded the culturing medium,replaced with serum-free medium, Interventing factors were added to the culture medium and the final volume was 1 ml,and GABA concentration was 4×10^-6mol/L,Bicuculine concentration was 1×10^-6mol/L,Forskolin concentration was 1×10^-3 mol/L,HCG concentration was 0.11U/ml.After twenty-four hours cultured,the supernatants were collected.2.Divide into groups:(1) Adding GABA(4×10^-6mol/L)to the culturing medium,and observating the effect of GABA on E₂ and P secretion by human luteinsing granulosa cells in vitro(n=8),divide into two groups:control group;GABA group(2) Adding Bicuculine(1×10^-6mol/L)to the culturing medium,and observating the effect of GABA on E₂ and P secretion under the influence of Bicuculine by human luteinsing granulosa cells in vitro(n=8),divide into four groups:control group; GABA group;Bicuculine group;GABA+ Bicuculine group.(3) Adding Forskolin(1×10^-3 mol/L) to the culturing medium,and observating the effect of GABA on E₂ and P secretion under the influence of Forskolin by human luteinsing granulosa cells in vitro(n=8),divide into four groups:control group; GABA group;Forskolin group;GABA+Forskolin group.(4) Adding hCG(0.1IU/ml) to the culturing medium,and observating the effect of GABA on E₂ and P secretion under the influence of hCG by human luteinsing granulosa cells in vitro(n=8),divide into four groups:control group;GABA group; hCG group;GABA+hCG group.3.Statistical methods to detect the concentration of estradiol and progesterone in supernatants by electrochemiluminescence technique.Comparing their variety of control group and treatment groups.Data were evaluated using SPSS13.0 software. Independment-sample T test was performed to analyse their variety.P＜0.05 was accepted as indicative of stastical significance.Results1.The GABA concentration of 4×10^-6mol/L could inhibit E₂ and P secretion by human luteinsing granulosa cells in vitro.2.Bicuculine concentration of 1×10^-6 mol/L can promote E₂ and P secretion by human luteinsing granulosa cells in vitro,and GABA can inhibit E₂ and P secretion under the influence of Bicuculine.3.Forskolin concentration of 1×10^-3 mol/L can promote E₂ and P secretion by human luteinsing granulosa cells in vitro,and GABA can inhibit E₂ and P secretion under the influence of Forskolin.4.HCG concentration of 0.1IU/ml can promote E₂ and P secretion by human luteinsing granulosa cells in vitro,and GABA can inhibit E₂ and P secretion under the influence of hCG.Summary1.GABA inhibit E₂ and P secretion by human luteinsing granulosa cells in vitro.2.the possible mechnism of GABA affect E₂ and P secretion by human luteinsing granulosa cells in vitro are mediated Through the GABA receptor on the membrane particles luteinsing granulosa cells in vitro.3.the possible mechnism of GABA affect E₂ and P secretion by human luteinsing granulosa cells in vitro are associated with the adenylate cyclase system;4.the possible mechnism of GABA affect E₂ and P secretion by human luteinsing granulosa cells in vitro are GABA and HCG with the corresponding receptor interaction may affect each other.PART TWO Effect of GABA on apoptosis by human ovarian granulosa cells in vitroMany studies reported that ovarian somatic cells and germ cells apoptosis were related to follicular atresia,and study on ovarian somatic cells apoptosis also focused on granulosa cells.ObjectiveTo invistigate GABA concentration in follicular fluid and how exogenous GABA affect human luteinized granulosa cells apoptosis in IVF cycles.Methods1 Forty barren patients were treated by IVF/ICSI-ET in southern hospital's reproductive center.They were less than thirty-five years old.Barren cause was fallopian tube factor or male factor.They were all at their first IVF/ICSI-ET cycle using down regulation long controlled hyperstimulation protocol.â‘ Granulosa cells collected:Centrifuging and separating luteinized granulosa cells,the isolated luteinized granulosa cells were divided into two groups to carry out in vitro for twenty-four hours,Discarded the culturing medium,replaced with serum-free medium,GABA were added to treat group,the final volume was 1 ml,and GABA concentration was 4×10^-6mol/L.Continue to cultivate twenty-four hours,discarded supernatants,and collected adherent cells to smear.â‘¡follicular fluids collected:collecting matural follicular fluid(≥15mm) on taking ovum day,and follicular fluids were centrifuged in centrifugal machine,then to collect the supernatant.Supernatant were put the refrigerator.2 Divide into groups:(1) Divided into two groups:Pregnancy group(n=17) and non-pregnancy group(n=23),GABA concentration were measured in follicular fluid of the two groups,and comparison the GABA concentration difference of two groups.(2) Divided into control group and GABA group,comparison apoptosis rates.3 To detect the concentration ofγ-gamma-aminobutyric acid in follicle fluids by humanγ-gamma-aminobutyric acid ELISA kit.Comparing their variety of two group.Data were evaluated using SPSS13.0 software.Independment-sample T test was performed to analyse their variety.P＜0.05 was accepted as indicative of stastical significance.Results1.The concentration ofγ-gamma-aminobutyric acid in follicle fluid in pregnancy group was lower than one of non-pregnancy group (680.398±11.279 vs 713.832±9.517) ng/ml,and the difference was stastical significance(P＜0.05).2.Apoptosis rates of human luteinized granulosa cells in GABA group was higher than one of in control group(20.08±2.44 vs 29.27±2.02)%,and the difference was stastical significance(P＜0.05). Summary1.We found that GABA concentration of human follicular fluid in non-pregrant group was higher than one of in pregrant group,and the difference was stastical significance2.We added GABA to human luteinized granulosa cells in vitro,and found that GABA could promote apoptosis rate of human luteinized granulosa cells in vitro,and the difference was stastical significance CONCLUSIONS1.The experiment using electrochemical luminescence detection kit E2 and P concentration, observing the effect of GABA on E₂ and P Secretions by Human Ovarian Granulosa Cells in Vitro,explore the possible mechanism of exogenous GABA effect on human luteinized granulosa cells secretion of estradiol and progesterone.The results suggested:â‘ GABA inhibit E₂ and P secretion by human luteinsing granulosa cells in vitro.â‘¡the possible mechnism of GABA affect E₂ and P secretion by human luteinsing granulosa cells in vitro are mediated Through the GABA receptor on the membrane particles luteinsing granulosa cells in vitro;are associated with the adenylate cyclase system;are GABA and HCG with the corresponding receptor interaction may affect each other.2.We used ELISA technique to detect GABA concentration of pregnancy group and non -pregnancy group in IVF cycles.The result was that the concentration of GABA in follicle fluid of non-pregnancy group was significantly higher than one of pregnancy group.And we used TUNNEL technique to detect the effect of exogenous GABA on human luteinized granulosa cell apoptosis,the result was that apoptosis rates of human luteinized granulosa cells in GABA group was higher than one of in control group.The experiment suggested that high concentrations of GABA may increase the rate of apoptosis of granulosa cells,thus affect the quality of oocytes,and reduce the pregnancy rate.