G蛋白偶联受体抑制剂与成人卵巢组织体外共培养通过抑制HIPPO-YAP信号通路促进始基卵泡募集

GPCRs inhibitor activates primordial follicles by inhibiting Hippo-YAP signaling pathway

目的本研究观察G蛋白偶联受体抑制剂溶血磷脂酸(LPA)及1-磷酸硝氨醇(S1P)与成人卵巢组织共培养后Hippo-YAP信号通路各元件及下游蛋白因子的表达及卵巢组织中卵泡发育相关标记的变化,探讨其通过Hippo-YAP信号通路调控始基卵泡募集的作用。方法收集中山大学附属第一医院2017年3月-2017年11月妇科手术切除的正常成人卵巢组织,通过WB、ELISA等方法检测分别经GPCRs抑制剂共培养、机械卵巢组织碎片化及其与Akt通路激活剂共培养结合使用后组织中通路各元件及下游蛋白因子的表达及培养液中卵泡发育相关指标的浓度,观察不同处理方式对Hippo-YAP通路抑制效应及对卵巢内卵泡募集的效果。结果(1)成人卵巢皮质组织使用5μmol/L LPA及5μmol/L S1P联合培养体系共培养8h左右可获得较明显的Hippo-YAP通路效应因子CCN2的表达促进效果(P<0.01);(2)正常成人卵巢皮质组织中,AKT信号通路激活剂共培养48 h后与LPA及S1P共培养8 h可获得与目前体外卵泡激活标准的“机械碎片化HIPPO-YAP信号通路抑制法+AKT信号通路激活剂共培养48 h”方案相接近的HIPPO-YAP信号通路主要元件YAP的磷酸化抑制及下游效应因子CCN2表达增加效应(P>0.5)。结论(1)GPCRs抑制剂LPA及S1P共培养后在人卵巢组织呈现Hippo-YAP通路抑制效应;(2)在体外培养体系中,人卵巢皮质组织以5μmol/L LPA及5μmol/L S1P组成的总浓度为10μmol/L的GPCRs抑制剂处理,培养24 h后可获得较佳的Hippo-YAP通路抑制效应,但Hippo-YAP信号通路抑制产生的促卵巢组织生长的效应在通路抑制效应后仍可能持续存在。

Objective To explore the effect of GPCRs inhibitors,L-α-Lysobisphosphatidic acids(LPA)and Sphingosine 1-phosphate(S1P),on the Hippo-YAP signaling pathway after co-cultured with human ovarian tissue in vitro and its role in the activation of primordial follicles in human ovarian tissue,eventually provide us with brand new data and therapeutic methods on female infertility related with follicle growth.Methods Normal adult ovarian tissues removed by gynecological surgery in the First Affiliated Hospital of Sun Yat-sen University from March 2017 to November 2017 were collected,and after regulating the Hippo-YAP signaling pathway with different methods including co-culture with GPCRs inhibitor,co-culture with PTEN inhibitor and PI3K activator and/or ovarian tissue fragmentation,we performed WB and enzyme-linked immunosorbent assay(ELISA)to analyze whether and how the different methods have different effectiveness on regulating the Hippo-YAP signaling pathway and the follicle growth.Results(1)The expression level of the main elements of HippoYAP signaling pathway and its downstream effect factor CCN2 were both down-regulated in human ovarian tissue(P<0.01).(2)In the in vitro culture system,we have achieved results suggesting the ideal culture duration may be 24 hours to achieve best inhibiting effect of Hippo-YAP signaling pathway.Using Akt signaling pathway activation group as negative control and combining Akt signaling pathway activation with tissue fragmentation group as positive control,the result suggested that different methods may achieve similar inhibiting effect on Hippo-YAP signaling pathway in human ovarian tissue.Conclusion(1)GPCRs inhibitors,LPA and S1P,inhibits the Hippo-YAP signaling pathway after co-cultured with human ovarian tissue.(2)By analyzing the main elements of Hippo-YAP signaling pathway,p-YAP/YAP and its downstream effect factor CCN2,as the evaluating indicator in the in vitro culture system,the GPCRs inhibitors showed the strongest inhibitory effect after 24 hours of co-culture in human ovarian tissue.3.Inhibition of Hippo-YAP signaling pathway in both ways including tissue fragmentation and co-culture with GPCRs inhibitors,combined with Akt signaling pathway activating,may achieve a similar inhibition level of Hippo-YAP signaling pathway.