摘要
不对称细胞分裂是指一个细胞分裂成两种不同发育潜能的细胞,是产生细胞多样性的一种基础方式。不对称分裂产生的两种类型的细胞在大小、形态和发育遗传物质上均有所不同。发育遗传物质在细胞内分布产生极性化,分裂平面将这些物质在子细胞将不均等分配,从而使得许多组织中产生两种不同发育潜能的细胞。
在所有种属中,卵子成熟都是一种极其典型的不对称分裂。许多不对称分裂细胞在分裂前需要在细胞内产生不对称性,也就暗示着需要极性建立分子的存在。其中一些不对称性在卵子受精前即已经存在。在鼠卵中,卵子经过连续两次不对称减数分裂产生两个小的极体和一个大的卵母细胞。减数分裂的极性是由第一次减数分裂纺锤体和第一极体的极性位置决定和形成的,接着由第二次减数分裂纺锤体和第二极体的极性位置来延续。这样,阻滞于第二次减数分裂中期的鼠卵母细胞已经成为了一个极性化了的细胞。鼠卵的极性反应在减数分裂纺锤体和极体的位置以及卵浆成分的不对称分布上。通常认为卵浆和卵膜的结构和分子极性是由成熟过程中减数分裂纺锤体的极性位置决定的。但哺乳动物卵子减数分裂过程中调节纺锤体不对称位置的分子机制仍旧不甚清楚。而不同种属间不对称分裂的调节机制存在高度的进化保守性。人们对单细胞生物和多细胞生物的细胞母源性因子的分布和功能展开了广泛的研究,我们可通过这些研究来推断和发现鼠卵不对称分裂的机制。
本实验主要研究三个进化高度保守的不对称分裂分子在鼠卵减数分裂和早期植入前胚胎的表达和功能,研究主要分为以下两块:
1.Dlg1在鼠卵减数分裂中的表达和功能研究
2.Epac和Rap1在鼠卵和植入前胚胎中的表达和功能研究
第一部分:Dlg1在鼠卵减数分裂中的表达和功能研究
一:鼠卵减数分裂过程中Dlg1的极性化分布
研究背景和目的:研究表明Discs large homolog 1(Dlg1)蛋白在果蝇神经母细胞细胞极性形成中起到重要的作用。在神经母细胞极性形成过程中,Dlg1参与调节两大保守蛋白Pins和Gαi的皮质位置的形成,而这两大保守蛋白在从昆虫乃至哺乳动物的研究中,都证实了在调节细胞不对称分裂和纺锤体与皮质间关系中起到中心性作用。但是Dlg1在哺乳动物卵子中参与极性调节的作用却未见相关报道,本实验研究Dlg1在鼠卵中的表达和其与细胞骨架的关系,从而为进一步功能研究奠定基础。
材料和方法:首先,我们用western-blot和RT-PCR验证Dlg1在鼠卵中的表达;其次用卵巢石蜡切片免疫组化方法揭示了Dlg1在卵巢的分布和表达;接着又研究了Dlg1在不同时期体内和体外卵子的亚细胞定位,并使用一些药物揭示了Dlg1和细胞骨架微丝微管之间的关系。
结果:western-blot半定量分析和RT-PCR表明GV期和MII期卵子均表达Dlg1蛋白,且MII期Dlg1含量高于GV期。且western-blot结果验证了抗体的特异性。在小鼠卵巢中,Dlg1呈弥漫性分布,生殖细胞和卵泡细胞均有表达,且Dlg1在颗粒细胞中的表达伴随着卵泡发育而增强;卵子中,Dlg1分布于各个时期未成熟卵子卵浆内。单细胞免疫荧光揭示了Dlg1在减数分裂不同时期的细胞内分布。GV期,Dlg1分布于整个卵浆,且在核膜周围表达明显增强;生发泡破裂后,Dlg1重新组装并与纺锤体共表达。在GV期阻滞的卵子中,Dlg1不会发生重组装,这说明生发泡破裂启动了Dlg1的重分布。且Dlg1与纺锤体的共存可被微管特异性药物诺考达唑所破坏,故Dlg1的纺锤体样分布呈微管依赖性。伴随着纺锤体和Dlg1的极性向动物极皮质区迁移,Dlg1在对侧植物极皮质区域也出现了一种特异性分布类型—散点颗粒组成的皮质下方月牙状结构,该结构伴随着纺锤体的迁移变得更加明显,直到第一极体排出MII期形成时这种分布变得最清晰,而这种结构可被微丝解聚剂细胞松弛素B所破坏,这样该结构的形成呈微丝依赖性。
结论:Dlg1存在于卵母细胞和周围颗粒细胞中,Dlg1可能形成参与颗粒细胞间的紧密连接。Dlg1可能亚细胞定位于内质网,鼠卵减数分裂过程诱导了Dlg1的极性分布,这种极性化分布呈多步骤性,受微丝和微管的调节。Dlg1分布与细胞骨架的密切关系,提示其可能参与或调节卵子极性形成,有待于进一步实验证明。
二:母源性缺乏Dlg1阻滞了减数分裂进程
研究背景和目的:RAN干扰技术在研究哺乳动物基因功能中是一种有效的工具。其中小发卡RNA更具有降低非特异基因干扰和增加干扰效率的优势。本实验用Dlg1基因特异性shRNA通过显微注射方法导入卵母细胞研究Dlg1在鼠卵减数分裂中的功能。
材料和方法:2对长度为21-nt的Dlg1 siRNA购自Ambion公司(属预合成基因,任意两条中保证一条干扰效率达70%以上)。终浓度分别为50μM和100μM的siRNA通过显微注射的方法导入GV期卵母细胞卵浆。每个卵子的注射量约为10pL。对照组注射等量的阳性(GAPDH)或阴性对照。注射完毕后,卵子在含dbcAMP的G1培养液中培养30min,然后转入IVM培养基中,并在4hr、8hr和18hr分别统计GVBD率和第一极体排出率。同时进行Real-time RT-PCR和免疫荧光分析。
结果:最初实验采用50μM浓度注射,结果两对siRNA均未对细胞周期进程产生影响。siRNA浓度换成100μM后,两对siRNA均可对Dlg1基因表达产生沉默,但ID160541效果较好且沉默率可达到70%,且18小时时沉默效率达到最高。4hr,8hr和18hr时,实验组GVBD率和第一极体排出率均明显低于阴性对照组,差别有统计学意义,不同组间GAPDH基因表达无差异。取实验组MI卵和MII行免疫荧光分析,结果多数卵子可观察到特异性分布消失和荧光强度较弱现象。
结论:Dlg1基因沉默效率呈时间和浓度依赖性,ID160541可达到最好干扰效率且干扰效率最高时间为注射后18小时。母源性缺乏Dlg1可影响和阻滞减数分裂的启动和进程。
第二部分:Epac和Rap1在鼠卵和植入前胚胎的表达和功能研究
一:Epac和Rap1在鼠卵和植入前胚胎的动态表达
研究背景和目的:cAMP是一种重要的第二信使,在卵子中通过激活蛋白激酶A(PKA)来调控减数分裂的启动。事实上PKA并不是cAMP的唯一靶蛋白,新的关于cAMP依赖性但PKA非依赖性通路研究表明cAMP/Epac/Rap1信号通路同样参与与许多类型细胞的功能行使,包括细胞分泌、细胞间粘附和连接、细胞凋亡、细胞增殖和分化等等。该通路在cAMP信号传导过程中,与PKA依赖性信号通路相互协调和补充共同扩大cAMP的作用范围而完成多种细胞功能的行使。然而该通路在鼠卵和胚胎中是否存在却未见相关报道。
方法:用小鼠卵巢石蜡切片免疫组化和RT-PCR方法证实了Epac和Rap1在卵巢和卵子内的表达,同时用单细胞免疫荧光方法分别分析了Epac和Rap1在卵子和胚胎内的分布。
结果:首先,利用小鼠卵巢石蜡切片免疫组化的方法证实了Epac和Rap1均同时表达于各级小鼠卵母细胞和周围卵泡颗粒细胞,RT-PCR方法验证了Epac和Rap1在mRNA水平的表达,单细胞免疫荧光结合共聚焦显微镜分析了Epac和Rap1在不同时期卵子和早期植入前胚胎的亚细胞分布特征。Epac伴随着鼠卵成熟过程,其分布也发生着特征性动态变化。GV期,Epac蛋白聚集成明显颗粒状散布在整个卵浆,呈圆点状,大小不一;生发泡破裂后,这种颗粒性分布变得更典型,分布颗粒更加密集且范围更广泛,大小一致;随后伴随着染色体向皮质方向移动,Epac蛋白颗粒性也同时呈现出极性分布,即只位于染色体对侧的皮质区域;第一极体排出后卵子发育至第二次减数分裂末期时,该颗粒性分布变得不明显,仅在卵浆中偶可见;卵子受精后发育至有明显原核期胚胎时,Epac分布呈弥散均匀状,特征性颗粒状分布消失。而作为Epac下游蛋白的Rap1在鼠卵和胚胎中的分布却完全不同。卵母细胞成熟过程中,从GV期到MII期,Rap1分布无明显变化,呈弥散均匀状遍布于整个卵浆中,分布上无明显特征性和极性;但对植入前胚胎(1细胞期至4细胞期)分析发现,Rap1在早期胚胎中分布发生明显变化,胞浆中出现较多的颗粒呈周边分布,与Epac在卵子中颗粒分布不同的是,Rap1颗粒较小,呈逗点状,大小均匀。而在以后的胚胎中,除了以上分布特征之外,可见散落融合在一起成簇的颗粒,分布在胞浆皮质区。
结论:Epac和Rap1存在于鼠卵减数分裂成熟过程及植入前早期胚胎中,Epac和Rap1在卵子和胚胎发育不同时期呈动态性分布,且分别具有一定的特征性,各不相同。研究提示鼠卵和植入前胚胎中可能存在cAMP/Epac/Rap1通路。
二:Epac和Rap1在鼠卵和植入前胚胎的功能分析
研究背景和目的:之前研究我们揭示了Epac和Rap1在鼠卵和胚胎中分别有特异性的表达,Epac在卵子成熟过程中分布存在特异性,但发育至MII期后该特异性消失;而Rap1的特异性分布开始于原核期胚胎。本实验基于以上实验分别研究Epac在卵子成熟过程和Rap1在胚胎发育过程对细胞周期进程的影响。
材料和方法:GV卵子和原核胚胎分别从5周龄B6D2F1未刺激卵巢和刺激周期输卵管中获得,Epac和Rap1抗体(1ml PBS中含200mg/ml IgG和0.1%NaN3和0.2%gelatin)分别显微注射于卵浆和原核期胚胎胞浆内。每个实验重复三次,每组每次注射30-40个卵子,所有实验中单个卵子或胚胎注射量约为10pl。对照组为等量的兔IgG(1ml PBS中含200mg/ml IgG和0.1%NaN3和0.2%gelatin),注射完毕后分别置于IVM和G1培养液中观察对细胞周期进程的影响,同时结合免疫荧光方法分析。
结果:Epac对卵子成熟的影响:抗体注射后3hr GVBD率实验组(71.6%,155/217)明显低于对照组(85.3%,91/107):注射后18hr实验组第一极体排出率(12.9%,28/217)明显低于实验组(67.2%,71/107)。注射后3hr实验组GV阻滞卵子免疫荧光分析显示卵浆内粗颗粒状减弱或分布消失,验证了抗体注射的效果。Rap1对早期植入前胚胎有丝分裂进程的影响:注射后24hr(2细胞胚胎)和48hr的胚胎情况(4细胞胚胎)实验组(99.3%136/137;98.5%,135/137)和对照组(100%83/83;100%,83/83)间无明显区别;但抗体注射后确实影响了胚胎发育的最终结局—囊胚形成率实验组(42.3%,58/137)明显低于对照组(80.7%,67/83)。注射后48hr实验组4细胞胚胎Rap1免疫荧光染色见胞浆内Rap1染色呈低信号,颗粒状分布基本消失。
结论:Epac可影响卵子减数分裂的细胞周期进程,而Rap对早期胚胎发育的有丝分裂细胞周期进程无明显影响,却影响了胚胎发育的最终结局。
Asymmetric cell division,in which a cell divides into two cells of different developmental potentials,is a fundamental means of generating cell diversity.An asymmetric division produces two cell types that differ in size,morphology,and/or the developmental determinants that they inherit.In the latter case,determinants are polarized within a cell and the division plane partitions these determinants unequally between the daughter cells,producing two cells with different development in many organisms.
In all organisms,oocyte maturation is an extreme example of asymmetric division.Many asymmetric divisions require the establishment of asymmetries in the cell prior to division,which implies the existence of polarity-estbalishing molecules. Some of these asymmetries are set up already in the oocyte,before fertilization.In the mouse,the female germ cells undergo two consecutive meiotic divisions with unequal cytokinesis thus forming two polar bodies at the animal pole of the egg.The polarity of meiotic divisions develops during the first meiotic metaphase spindle and the polar location of the first meiotic metaphase spindle and the polar location of the first polar body,which is followed by the polar localization of the second meiotic spindle and of the second polar body.As a result,the mouse secondary oocyte,arrested in the metaphase of the second meiotic division,is already a polarized cell.The polarity of the oocyte is reflected in the localization of meiotic spindle and polar body and the asymmetrical distribution of cytoplasm.It is believed that the structural and molecular polarity of the oocyte cytoplasm,and its oolemma are caused by the polar positioning of the meiotic spindle during maturation.The mechanism that in mammals regulates asymmetrical positioning of the spindle during meiotic division of the oocyte remains elusive.But the mechanism between different organisms is largely evolutionarily conserved.The cellular distribution of maternal factors and their functions are usually studied in single cell organisms and multi-cell epithelium.
Accordingly,this study was designed with the purpose to disclose the role of two conserved genes or pathway in oocyte maturation and early preimplantation embryos, which is divided into the following two parts:
1.Expression and functional analysis of the cAMP/Epac/Rap1 pathway at oocytes and early perimplantation embryos.
2.Expression and functional analysis of Dlg1 during oocyte maturation.
PART ONE:EXPRESSION AND FUNCTIONAL ANALYSIS OF DLG1 IN MOUSE OOCYTES
Ⅰ:MEIOTIC MATURATION INDUCES ASYMMETRIC DISTRIBUTION OF DLG1 IN MOUSE OOCYTES.
Background and Purpose:Discs large homolog 1(Dlg1) proteins had been demonstrated to play an important role in cell polarity in Drosophila neuroblasts, where they induced the cortical localization of two conserved proteins(Pins and Gαi). These two cortical proteins play central role in regulating asymmetric cell division and spindle-cortex interactions from worms to mammal.But Dlg1's role in mammal oocyte has never been investigated.Here we aim to reveal the distribution of Dlg1 in different stages of oocytes and find its relationship with cytoskeleton.
Materials and Methods:Firstly,we used Western-blot and RT-PCR to identify Dlg1's existence at different stages of mouse oocytes.Next we used immunohistochemistry to disclose its distribution in ovary.Oocytes collected in vitro or matured in vivo were fixed for immunocytochemistry to demonstrate the localization of Dlg1.Finally,we disclosed the relationship between the distribution of Dlg1 and cytoskeleton by adding specific drugs to the maturation medium at different time.
Results:Western-blot and RT-PCR analysis showed that Dlg1 was present in GV and MII-stage oocyte,and its quantity seemed doubled during meiotic maturation.For the first time,we showed that Dlg1 was expressed in both germ and follicle cell through different stages of oocyte development from primordial follicle(＜80μm) to Graafian follicle(＞380μm).For the oocytes,Dlg1 staining distributed throughout the cytoplasm and various stages of oocytes.It appeared that the intensity of Dlg1 staining got higher in follicle cells as the progression of oogenesis.And we investigated thoroughly its spatial-temporal subcellular location during oocyte maturation.Firstly,Dlg1 was evident around the GV membrane and uniformly distributed in the cytoplasm.Dlg1 was restructured to colocalize with spindle at the time of GVBD.GVBD was essential for the initiation of Dlg1 reorganization,since Dlg1 structure did not change in GV-arrested oocyte.Dlg1 reorganization was also prevented by the microtubule inhibitor nocodazole.Dlg1's redistribution at GVBD was therefore MTs(microtubules)-dependent and cell cycle-dependent.Then the colocaliztion of Dlg1 with spindle began to migrate to the oocyte cortex,companied by emerging network of Dlg1 at the vegetal cortical.Distinctive cresent vegetal cortical clusters of Dlg1 were formed close to the time of Pb1(polar body 1) extrusion and were ever evident in MII oocytes.Formation of the characteristic Dlg1 clusters was prevented by the deploymerisation of microfilaments.
Conclusions:These experiments revealed that Dlg1 exists at different stages of oocytes and follicle cells,Dlg1 may involve in the regulation of tight junction of follicle cells.Mouse oocyte meiotic maturation induced the polarization of Dlg1 and this polarized action is highly related with cell cytoskeleton.Dlg1 may involve in the regulation and establishment of oocyte polarity.
Ⅱ:MATERNAL DEFICIENCY OF DLG1 BLOCKS MEIOTIC MATURATION IN MOUSE OOCYTES.
Background and Purpose:RNA interference(RNAi) is an effective tool for studying gene function in oocytes.Short hairpin RNAs(shRNA) has the advantage of reducing the potential for off-target interference and increase RNAi specificity in oocytes.
Materials and Methods:Two couple(ID 160540 and ID 160541) of predesinged annealed 21-nt shRNA for Dlg1 was chemically synthesized by Ambion company. shRNA in a final concentration of 50μM and 100μM was microinjected into the cytoplasm of GV stage oocytes.Each oocyte was injected about 10 pL of siRNA.The control group was injected with same amount of positive(GAPDH) control and negative control.After microinjection,the oocytes were incubated in G1 medium containing 100μg/ml cAMP for 30min and then washed thoroughly with IVM medium.The oocytes were then cultured in IVM medium for the evaluation of GVBD and PB1 extrusion after 4 hr,8 hr and 18 hr respectively.The oocytes were collected for real-time RT-PCR to measure the mRNA levels and immunocytochemistry and confocal analysis at the same time.
Results:In the first place,a concentration of 50μM was used for microinjection,both of the two couples of shRNA microinjected have no effect on cell cycle progression of oocyte maturation.So we increased the concentration of shRNA to 100μM.The two siRNA sequences all have silencing effect on Dlg1,but ID 160540 was better and its inhibitory rate was 70%.The most efficient time was 18 hr post microinjection.In 4 hr,8 hr and 18 hr,the rate of GVBD and Pb1 emission of Dlg1 siRNA injected were significantly lower than the negative control group.There was no difference between each group in GAPDH gene.
Conclusions:The effect of Dlg1 siRNA in mouse oocyte was time-and concentration-dependent.The most efficient siRNA sequence was ID 160540 and the time was 18 hr.Maternal deficiency of Dlg1 blocked the oocyte maturation progress.
PART TWO:EXPRESSION AND FUNCTIONAL ANALYSIS OF EPAC AND RAP1 IN OOCYTES AND PREIMPLANTATION EMBRYOS.
Ⅰ:DYNAMIC EXPRESSION OF EPAC AND RAP1 IN MOUSE OOCYTES AND PREIMPLANTATION EMBRYOS
Background and Purpose:cAMP is an important second messenger that has long been recognized to control the initiation of meiosis through activation of Protein Kinase A(PKA) in oocytes.However,PKA is not the only target for cAMP.Recent work in cAMP-dependent and PKA-independent pathways suggests that Rap1 (Ras-related protein-1) is activated through its Epac proteins(exchange proteins directly activated by cAMP),cAMP responsive guanine exchange factors(GEFs) involved in various cellular processes.Evidence is accumulating that Rap1 is clearly linked to actin dynamics and functions in the spatial and temporal control of cell polarity.The aim of this study was to reveal the existence of an Epac and Rap1 in mouse oocytes and embryos.
Materials and Methods:Five-week-old B6D2F1 mouse ovaries were fixed for immunohistochemistry.Different stages of oocytes and early preimplantation embryos were used for RT-PCR and immunocytochemistry.
Results:Results obtained indicate that Epac and Rap1 have different dynamic subcellular localizations and expression patterns in oocytes and embryos.For Epac,at germinal vesicle stage,Epac was first seen as small accumulations scattered in the cytoplasm;after germinal vesicle breakdown,a network of small clusters,distributed uniformly throughout the cytoplasm,then became evident;but at the meiosisⅡstage, the accumulations appeared to become reduced and spread out in the ovoplasm;at one-cell stage,the clusters disappeared and Epac became uniformly distributed throughout the embryos.But Rap1 dispersed uniformly as small punctuations throughout the cytoplasm during the progress of oocyte maturation.When developed into one-cell stage embryos,the small dots aggregated into visible clusters of particles that mainly occupied the cortical region of the blastomeres.This phenomenon was evident even in two-cell and four-cell stage embryos and persistent in subsequent early preimplantation embryos.
Conclusions:These results indicate for the first time that the Eapc and Rap1 do exist and have differential dynamic subcellular distribution in mouse oocytes and embryos, which in turn suggests multiple and specific functions during oocyte maturation and embryo development.
Ⅱ:FUNCTIONAL ANALYSIS OF EPAC AND RAP1 IN MOUSE OOCYTES AND EMBRYOS
Background and Purpose:In a former experiment,we demonstated that Epac and Rap1 have differential dynamic expression pattern in oocytes and embryos.According to the earlier experiment,Epac had a unique distribution during oocyte maturation but this characteristic expression was lost when entering MII stage.And Rap1 got its typical expression only beginning from pronuclear-stage embryos.So in the present study,we investigated the functional role of Epac and Rap1 in oocytes and embryos based on their unique subcellular locations.
Materials and Methods:GV oocytes and pronuclear stage embryos were recovered from the ovaries and oviducts of primed 5-week-old B6D2F1 female mice.Epac and Rap1 antibodies(200 mg/ml in 1ml PBS containing 0.1%NaN3 and 0.2%gelatin) were microinjected into the cytoplasm of GV oocytes and 1cell stage embryos respectively.Each experiment was repeated three times,and 30-40 oocytes per group per experiment were used.A microinjection volume of about 10 pl per oocytes or embryos was used in all the experiments.The same amount of rabbit IgG(200 mg/ml in 1ml PBS containing 0.1%NaN3 and 0.2%gelatin) was injected as control.The oocytes and embryos were then cultured in IVM and G1 medium respectively for the evaluation of cell cycle progression and immunocytochemistry and confocal analysis at the same time.
Results:We investigated the effects of Epac on oocyte maturation by antibody microinjection.The GVBD rate of oocytes 3hr after antibody injection(71.6%, 155/217) was significantly lower than that of the IgG-injected control group(85.3%, 91/107).The rate of the first polar body extrusion 13hr after antibody injection (12.9%,28/217) was significantly lower than that of the IgG-injected control group (67.2%,71/107).To determine importance of Rap1 in early embryo mitotic progression,anti-Rap1 antibody was microinjected into the cytoplasm of pronuclear-stage embryos and cleavage events were assessed in 24-hr intervals. Embryo development(99.3%,136/137) had not significant difference past the two-cell stage by 48 hr of culture compared to controls(100%,83/83).But the antibody microinjection did significantly compromise the outcome of blastula formation(42.3%,58/137) for a prolonged culture compared with control(80.7%, 67/83).
Conclusions:Epac and Rap1 do have differential effect on cell cycle progression during oocyte maturation and preimplantation embryo development.Epac present in GV oocytes plays an important role in the process of GVBD and first polar body extrusion.Rap1 may have no effect on the development of early stages of embryos but is responsible for outcome of blastula formation.
引文
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