激活素—抑制素—卵泡抑素系统在卵母细胞成熟过程中的影响机制及其在未成熟卵母细胞体外成熟技术中的应用

发布时间：2018-04-10 14:49

  本文选题：激活素 + 抑制素　； 参考：《中国协和医科大学》2007年博士论文

【摘要】： 目前人类尚无法完整揭示卵泡生长发育过程中的调控机理，主要原因在于对卵巢内部因子作用机制了解不清。激活素-抑制素-卵泡抑素(activin-inhibin-follistatin，ACT-INH-FS)系统是最重要的卵巢内部因子之一，其卵巢内部的旁自分泌作用机制有待阐明。 近年来人类未成熟卵母细胞的体外成熟(in vitro maturation，IVM)技术在不育症治疗中显示出重要的临床价值。但其妊娠率、种植率以及自然流产率尚不理想，主要原因在于尚未建立能够使未成熟卵母细胞同步获得细胞核和细胞浆成熟的培养体系。ACT-INH-FS作为卵泡体内成熟微环境中的重要因子，可能也是IVM培养体系中不可或缺的组分。 本研究目的是通过以下三方面内容，探讨激活素-抑制素-卵泡抑素系统在卵母细胞成熟过程中的旁分泌作用机制及其在人类不成熟卵母细胞体外成熟技术中的应用价值。 一、激活素-抑制素-卵泡抑素系统对卵母细胞成熟及胚胎发育潜能的影响 二、激活素-抑制素-卵泡抑素系统卵巢局部生理作用的阶段性与交互性 三、卵母细胞体外成熟培养体系的安全性相关研究 第一部分激活素-抑制素-卵泡抑素系统对卵母细胞成熟及胚胎发育潜能的影响 目的：探讨激活素-抑制素-卵泡抑素系统在小鼠生发泡(GV)期卵母细胞体外成熟及早期胚胎发育中的的影响机制。方法：3-4周龄昆明系雌性小鼠腹腔注射PMSG后48小时，获取GV期卵母细胞随机分入以下八组。对照组采用HTF+10％SPS；试验组分别为：100ng／ml ACTA；100ng／mlACTA+100ng／mlFS；100ng／mlINHA；100ng／mlINHA+100ng／mlFS；100ng／mlACTA+INHA；100ng／mlACTA+100ng／mlINHA+100ng／mlFS；100ng／ml FS。GV期卵母细胞体外培养16-18小时后，MⅡ期的卵母细胞进行体外受精，观察ACTA-INHA-FS对GV期卵母细胞生发泡破裂(GVBD)、第一极体(PB1)排出及受精率、卵裂率、囊胚形成率的影响。使用MitoTracker RED荧光探针对ACTA组以及ACTA+INHA合用组MⅡ期卵母细胞内活性线粒体数目及分布进行检测。结果：(1) ACTA、ACTA+INHA、单独使用FS均可显著促进GV期卵母细胞GVBD发生率(P＜0.05)。(2) ACTA、ACTA+INHA、INHA均可显著促进MⅡ期卵母细胞发生率(P＜0.05)，(3) INHA组受精率低于对照组(P＜0.05)，其余各组与对照组相比，，受精率无明显差异(P＞0.05)。(4) ACTA、INHA、ACTA+INHA组8细胞及囊胚形成率均显著高于对照组(P＜0.05)。(5) FS可以特异性拮抗ACTA的促进作用，但不能中和ACTA与INHA的协同作用(P＞0.05)。(6)单独使用FS组囊胚形成率高于对照组(40％vs 24.7％)，但差异尚不具有统计学意义(P=0.051)。(7)体外成熟后的MⅡ期卵母细胞内线粒体形态可以分为以下三类：周边型、半周型、弥散型，ACTA组以及ACTA+INHA合用组半周型以及弥散型的比例明显高于对照组，整体分布存在显著性差异(P＜0.01)结论：(1) ACTA、INHA对GV期卵母细胞体外成熟及胚胎发育潜能具有显著的促进作用，并可以被FS所拮抗。(2)在ACTA／INHA缺乏或不足的条件下，FS可出现ACTA样作用，促进卵母细胞成熟。(3) ACTA-INHA对体外成熟卵母细胞核质成熟同步化具促进作用。 第二部分激活素-抑制素-卵泡抑素系统旁自分泌作用的阶段性及交互性 目的：探讨生理周期中LH峰前后ACTA／INHA对GV期卵母细胞体外成熟的不同影响；激活素-抑制素-卵泡抑素系统与EGF的相互作用方式。方法：3-4周性成熟前小鼠分为两组，一组给予PMSG超排卵48小时后取卵，另一组予PMSG超排卵48小时后继续给予HCG10U 2小时后取卵。HCG刺激(HCG+)组和HCG未刺激(HCG-)组的GV期卵丘-卵母细胞复合物(COC)，在含有100ng／mlACTA+100ng／mlINHA培养液中体外培养14-18小时，MⅡ期的卵母细胞进行体外受精，观察ACTA-INHA-FS对GV期卵母细胞生发泡破裂(GVBD)、第一极体(PB1)排出及受精率、卵裂率、囊胚形成率的影响。GV期卵母细胞在含10ng／ml EGF及10ng／ml EGF+100ng／mlFS的培养液中培养16-18小时后，MⅡ期的卵母细胞进行体外受精，比较生发泡破裂(GVBD)、第一极体(PB1)排出及受精率、卵裂率、囊胚形成率的差别。结果：(1) HCG(+)组COC经体外成熟培养14小时后，卵丘细胞团扩散比例明显高于HCG(-)组(81.5％vs5.1％)。(2) HCG(+)组与HCG(-)桑椹胚形成率以及囊胚形成率存在非常明显差异(P＜0.01)。(3) FS可以显著抑制EGF对PB1排出率的促进作用(P＜0.01)但不能抑制EGF对胚胎发育的促进作用(P＞0.05)结论：(1) LH峰后的数小时内是卵母细胞成熟发育、维持核浆成熟同步化的关键时期。(2) LH峰值前后ACTA／INHA对GV期卵母细胞体外成熟的影响不同，LH峰后ACTA／INHA对早期胚胎发育的促进作用消失。(3)胞核成熟与胞浆成熟存在不同的调控途径；在核成熟的调控机理中，ACT系统是EGF的下游作用通路。。 第三部分卵母细胞体外成熟培养体系的安全性相关研究 目的：通过对MⅠ期卵母细胞骨架的激光共聚焦研究探讨体外成熟卵母细胞纺锤体、染色体结构异常与非整倍体发生率的关系；通过对MⅡ期卵母细胞染色体核型的检测评价激活素A-抑制素A用于卵母细胞体外成熟培养体系的安全性。方法：(1) GV期卵母细胞体外培养7小时，获取MⅠ期卵母细胞作为体外成熟组，体内发育的MⅠ期卵母细胞作为对照组。经免疫荧光染色，共聚焦荧光显微镜观察纺锤体形态、染色体分布。(2) GV期卵母细胞随机分入以下两组：对照组采用HTF+10％SPS；试验组采用100ng／mlACTA+INHA；体外培养16-18小时后，选择MⅡ期卵母细胞进行染色体核型分析。结果：(1)体外成熟组纺锤体结构异常以及染色体分布异常的比例均明显增高(34.6％vs 7.1％；26.9％vs 0；P＜0.01)。但成熟后的MⅡ期卵母细胞非整倍体发生率(0／31)并无增高。(2) ACTA+INHA组非整倍体发生率为7.1％，与对照组相比非整倍体发生率无显著增高(P＞0.05)。结论：(1)不成熟卵体外培养过程中存在纺锤体结构以及染色体分布的异常；卵母细胞可通过自检方式阻止结构异常的细胞进一步分裂。(2) ACTA-INHA不增加体外成熟卵母细胞的非整倍体发生率。 激活素-抑制素-卵泡抑素系统在卵母细胞成熟过程中的影响机制相关研究在国内尚无先例。国外的相关研究大多数限于对核成熟的调控作用方面，3篇关于牛的研究分别观察了ACTA或INHA对胚胎发育潜能的影响，但结果不甚一致。本研究首次将ACT-INH-FS作为一个系统，较以往研究更为全面地探讨其对卵母细胞成熟的调节机理，首次借助对卵母细胞超微结构的观察证实了其对胞浆成熟的促进作用。并探讨了激活素系统生理作用的阶段性和交互性，及其用于IVM培养体系的安全性，这些内容及结论亦未见相关报道。
[Abstract]:At present , the mechanism of regulation and regulation in the growth and development of follicles is not fully revealed . The main reason is that the mechanism of the internal factors of the ovary is not clear . The activin - statin - follistatin ( ACT - INH - FS ) system is one of the most important internal factors of the ovary , and the mechanism of paratual self - secretion in the ovary needs to be clarified .


In recent years , the in vitro maturation ( IVM ) technique of human immature oocytes has shown an important clinical value in the treatment of infertility , but its pregnancy rate , implantation rate and spontaneous abortion rate are not ideal .


The purpose of this study was to explore the mechanism of the parasecretory action of the activin - statin - follicle - inhibiting system in the maturation of oocytes and its application in the in vitro maturation of human immature oocytes by the following three aspects :


Effects of Activin - Inhibin - follicle - statin on the maturation and embryonic development potential of oocytes


2 . Phase and Interactivity of the Local Physiological Function of Activin - Inhibin - follicle Suppressor System in the Ovary


Study on the safety of mature culture system of oocytes in vitro


Effects of the first part of activin - statin - follicle - inhibiting system on oocyte maturation and embryonic development potential


Objective : To investigate the mechanism of activin - statin - follicle - inhibiting system in vitro maturation and early embryonic development of mouse oocytes . Methods : Forty - eight hours after intraperitoneal injection of PMSG into Kunming female mice , the following eight groups were randomly divided into eight groups : HTF + 10 % SPS ;
The experimental groups were 100 ng / ml ACTA , respectively .
100 ng / ml ACTA + 100ng / ml FS ;
100ng / ml INHA ;
100ng / ml INHA + 100ng / ml FS ;
100 ng / ml ACTA + INHA ;
100 ng / ml ACTA + 100ng / ml INHA + 100ng / ml FS ;
Results : ( 1 ) ACTA , ACTA + INHA and INHA could significantly promote the incidence of GVBD in GV phase ( P < 0.05 ) . ( 1 ) ACTA and INHA promoted the maturation and development potential of oocytes in vitro and could be antagonized by FS . ( 2 ) The ACTA - INHA promoted the maturation of oocytes and promoted the maturation of oocytes .


Phase and Interactivity of the Second Part of Activin - Inhibin - follicle Suppressor System in the Presence of Self - Secreting


Objective : To investigate the effect of ACTA / INHA on the maturation of GV oocytes in vitro before and after LH peak .
( 2 ) The effect of ACTA - INHA - FS on the growth of oocytes in GV stage was significantly higher than that in HCG ( - ) group ( 81.5 % v5.1 % ) .
In the mechanism of nucleus maturation , the ACT system is the downstream pathway of EGF .


Study on the safety of the third part oocyte in vitro maturation culture system


Objective : To investigate the relationship between chromosome structural abnormalities and the incidence of non - euploid in vitro maturation of oocytes in vitro .
Methods : ( 1 ) The oocytes were cultured in vitro for 7 hours in vitro . The oocytes were cultured in vitro for 7 hours in vitro .
100 ng / ml ACTA + INHA was used in the test group .
Results : ( 1 ) The abnormal structure of spindle structure and the abnormal distribution of chromosome distribution in vitro were significantly higher than that in the mature group ( 34 . 6 % vs 7.1 % ) .
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