线粒体与卵母细胞和早期胚胎发育

在哺乳动物成熟卵母细胞和早期胚胎中,线粒体是最独特且数量最多的细胞器,其不仅仅是产生ATP的细胞器,还参与许多重要的功能。哺乳动物卵母细胞和早期胚胎发育过程中伴随着线粒体诸多特性变化,如线粒体DNA拷贝数、mtDNA转录、线粒体超微结构、线粒体空间分布和线粒体膜电位。上述特性变化可影响卵母细胞和早期胚胎发育,甚至可导致卵母细胞和早期胚胎发育障碍。     

人成熟卵母细胞玻璃化冷冻技术的临床应用

目的探讨玻璃化冷冻技术应用于人成熟卵母细胞冷冻保存的临床效果。方法使用开放式载体cryotop进行人成熟卵母细胞的玻璃化冻存,对10例患者的冷冻卵子复苏后进行卵母细胞浆内单精子（ICSI）注射后行胚胎移植,观察复苏卵母细胞受精、卵裂、胚胎发育及临床妊娠情况。结果 10例患者共86枚玻璃化冻存的成熟卵母细胞复苏后77枚存活,69枚正常受精,获得65枚胚胎,共移植17枚胚胎,3例患者获临床妊娠,其中2例双胎妊娠。结论玻璃化冷冻技术是有效地保存人成熟卵母细胞的方法,有一定的临床应用价值。     

父源基因组重编程中组蛋白变体的作用

卵母细胞具有重编程精子基因组以确保胚胎正常发育的能力。精子入卵后,父源基因组会经历组蛋白替换鱼精蛋白,全基因组去甲基化等过程,从而启动胚胎发育。组蛋白H3的变体H3.3可以替换核小体中的典型组蛋白H3.1和H3.2,从而修饰染色质结构和影响基因表达。在早期胚胎发育过程中H3.3的缺失将导致染色体的过度凝集和错误分离。我们综述了组蛋白变体H3.3及其分子伴侣在精子发生、受精和早期胚胎发育中的作用,特别是H3.3对父源基因组重编程的重要性,这对理解受精后全能性合子的形成及着床前发育具有重要意义。     

体外成熟卵母细胞受精方式及发育能力的影响因素

目的 探讨体外成熟卵母细胞的受精方式及影响其胚胎发育的因素.方法 收集本院2008年7月至12月因男性因素需进行卵母细胞胞浆内单精子注射术(ICSI)助孕的135对夫妇未成熟的卵母细胞(DO)354枚,置入P1培养体系中观察培养20～24、26～30、36～40 h后的成熟情况.去除了退化的成熟卵母细胞,随机分为常规体外受精(IVF)和ICSI组,比较2组的正常受精率、卵裂率、第3天优质胚胎率及各组优质胚胎和非优质胚胎的患者年龄、不孕年限、促排时间及卵母细胞体外成熟时间.结果 卵母细胞体外成熟率为82.5%(292/354),IVF组正常受精率56.5%(70/124)低于ICSI组69.9%(107/153)(P<0.05).两组卵裂率和优质胚胎率差异无统计学意义(P>0.05).2组组内优质胚胎的患者年龄、体外成熟时间均小于非优质胚胎(P<0.05);不孕年限及促排时间差异无统计学意义(P>0.05).结论 ICSI能够提高体外成熟卵母细胞的受精率.体外成熟卵母细胞具有常规受精的能力,第3天胚胎质量与ICSI相似.患者年龄和卵母细胞体外成熟时间是体外成熟卵母细胞发育能力的主要影响因素.     

ICSI术治疗IVF受精异常周期的实验室数据分析

目的探讨由于受精障碍、异常受精或低受精指征行ICSI术患者的卵母细胞受精情况及胚胎发育情况。方法回顾性分析2009年1月至2010年12月,在本中心由于受精障碍、异常受精或低受精接受ICSI助孕治疗的45对夫妻为研究组,2010年1月一2010年12月由于重度少弱精接受ICSI的137例,为对照组。结果受精障碍、异常受精或低受精组的正常受精率和优质胚胎率明显低于重度少弱精组。结论ICSI能够改善受精障碍组的受精情况,但是正常受精的数量和胚胎发育潜能比较低。     

Ca2+载体A23187对受精失败人类成熟卵母细胞的补救激活作用

目的:观察Ca²⁺载体A23187对受精失败人类成熟卵母细胞的补救激活及激活后的胚胎发育情况。方法:收集常规体外受精(IVF)及卵浆内单精子注射(ICSI)后24h仍无受精征象的成熟卵母细胞作为研究对象,5μmol/LCa²⁺载体A23187中处理5min,观察第二极体排出及原核形成情况,激活后卵母细胞在体外继续培养2d。结果:IVF组及ICSI组卵母细胞激活率分别为64.9%(24/37)和73.2%(30/41)。ICSI组受精失败卵母细胞激活后主要表现为二极体二原核(2PB+2PN)(80%,24/30),而IVF组仅有20%的被激活卵母细胞表现为2PB+2PN,两组间差异具极显著(P＜0.01)。31个2PN卵母细胞继续培养,25个发生分裂,11个发育到2-4细胞,8个发育到4-8细胞,6个发育到8-细胞以上阶段。结论:Ca²⁺载体A23187能够有效地激活ICSI后受精失败卵母细胞恢复受精并继续发育成胚胎。     

硫氧还蛋白过氧化物酶Ⅵ在小鼠卵巢、输卵管和子宫中的分布

目的探讨硫氧还蛋白过氧化物酶VI(PrxVI)卵母细胞生长发育和早期胚胎发育中的可能作用。方法用免疫组织化学方法观察6～8周雌性昆明小鼠卵巢、输卵管和子宫PrxVI的分布。结果在卵巢中,PrxVI免疫阳性见于卵巢卵母细胞和黄体;在输卵管中,输卵管上皮呈PrxVI免疫阳性反应;在子宫中,PrxVI免疫阳性反应见于子宫内膜上皮、腺上皮和子宫内膜基质细胞。结论PrxVI的定位分布提示其可能参与小鼠卵母细胞生长发育和卵母细胞成熟过程的调节,并为受精卵和早期胚胎发育提供抗氧化的良好微环境。     

早期卵裂胚胎的发育和妊娠结局

目的探讨胚胎早期卵裂与胚胎发育速度、胚胎质量、临床妊娠结局之间的关系。方法这项回顾性研究对2012年8月至2013年5月共690个IVF/ICSI周期进行了比较分析。试验组于受精后25-27h观察早期分裂情况,将胚胎分为早期分裂胚胎,原核破裂胚胎和2原核合子期胚胎。胚胎发育至受精后66-68 h(第3天)对胚胎进行形态学评级,根据移植胚胎中早期卵裂胚胎的数目分为三组:2 EC组(2枚早期卵裂胚胎),2 Non-EC组(2枚非早期卵裂胚胎),混合组(1枚非早期卵裂胚胎和1枚早期卵裂胚胎)。结果早期卵裂胚胎发育速度要快于原核破裂胚胎和2原核合子期胚胎,而且早期卵裂胚胎和原核破裂胚胎的优胚率显著高于2原核合子期胚胎。2 EC组、2 Non-EC组和混合组三组的临床妊娠率和种植率无统计学差异。早期卵裂观察组与非早期卵裂观察组之间的临床妊娠率和种植率也没有显著差异。结论虽然早期卵裂与胚胎发育速度和优胚率有着密切的关系,但形态学评分均优的胚胎优先选择早期卵裂的移植策略尚不能明显改善临床妊娠结局。     

小鼠胚胎培养在新建胚胎实验室的质量控制研究

目的通过小鼠胚胎的生长情况,对新建体外受精(IVF)实验室的质量控制进行验证,为人类辅助生殖技术的开展提供理论依据。方法将体内受精与IVF方式得到的2细胞期胚胎放入不同培养箱中培养,观察胚胎生长情况。结果进行了15个周期的体内受精体外培养实验,共获得225个2细胞及以上胚胎,培养后形成囊胚204个囊胚,囊胚率为90.7%。进行了15个周期的体外受精实验,共获得315个卵母细胞。体外受精后集合培养,获得289个2细胞。培养后形成囊胚229个囊胚,囊胚率为79.3%。两组间差异有统计学意义(P0.05)。结论小鼠卵母细胞体内受精和体外受精后均有高的成胚率,体内受精后进行体外培养优于体外受精后培养,鼠胚实验可对新建胚胎培养室的培养体系进行评估。     

第二极体数目与受精结局及胚胎发育潜能的相关性

目的 探讨第二极体数目与卵母细胞受精结局和胚胎发育潜能之间的关系.方法 根据受精后5 h内卵母细胞极体数目的 不同分为1个极体组(1PB组)、2个极体组(2PB组)、3个极体组(3PB组)和4个以上极体组(≥4PB组).分别统计各组的正常/异常受精率(2PN率,1PN和3PN率)、优质胚胎率(优胚率)、移植胚胎所占比例...     

NuMA和γ-tubulin在核移植克隆胚胎中的动态变化和作用

核有丝分裂器蛋白(NuMA)和γ-微管蛋白(γ-tubulin)在卵母细胞成熟、受精和核移植胚胎早期发育过程中起重要作用。供体细胞γ-tubulin和NuMA参与了核移植克隆胚有丝分裂纺锤体的组装,NuMA主要参与了纺锤体极的装配和维持,而供体中心体调节核移植胚最初纺锤体的形成。核移植重构胚中心体结构异常经常伴随染色体异常排列、纺锤体结构异常,而中心体分布异常经常伴随异常卵裂。     

The nuclear mitotic apparatus (NuMA) protein: localization and dynamics in human oocytes, fertilization and early embryos

The oocyte's meiotic spindle is a dynamic structure that relies on microtubule organization and regulation by centrosomes. Disorganization of centrosomal proteins, including the nuclear mitotic apparatus (NuMA) protein and the molecular motor complex dynein/dynactin, can lead to chromosomal instability and developmental abnormalities. The present study reports the distribution and function of these proteins in human oocytes, zygotes and early embryos. A total of 239 oocytes, 90 zygotes and discarded embryos were fixed and analyzed with confocal microscopy for NuMA and dynactin distribution together with microtubules and chromatin. Microtubule-associated dynein-dependent transport functions were explored by inhibiting phosphatase and ATPase activity with sodium-orthovanadate (SOV). At germinal vesicle (GV) stages, NuMA was dispersed across the nucleoplasm. After GV breaks down, NuMA became cytoplasmic before localizing at the spindle poles in metaphase I and II oocytes. Aberrant NuMA localization patterns were found during oocyte in vitro maturation. After fertilization, normal and abnormal pronuclear stage zygotes and embryos displayed translocation of NuMA to interphase nuclei. SOV treatment for up to 2 h induced lower maturation rates with chromosomal scattering and ectopic localization of NuMA. Accurate distribution of NuMA is important for oocyte maturation, zygote and embryo development in humans. Proper assembly of NuMA is likely necessary for bipolar spindle organization and human oocyte developmental competence.     

Mitochondrial distribution and function in oocytes and early embryos

Active mitochondria relocate during oocyte maturation or fertilization in several species. Detailed studies with hamster oocytes and early embryos reveal a pattern of active mitochondria migrating to surround the pronuclei to form a pattern that persists through the early cleavage stages. Although the functional significance of this relocation is unknown, it appears to be an important part of normal development in hamsters. Treatments that disrupt embryo development in vitro (such as the presence of inorganic phosphate or alteration of intracellular pH) also disrupt the normal pattern of mitochondrial distribution. Active mitochondria also reorganize during maturation in bovine oocytes and during fertilization in rhesus monkey oocytes. Examination of these changes in mitochondrial organization may provide insights into the regulation of normal embryo development and might serve as predictors of oocyte or embryo developmental competence.     

线粒体能量代谢和DNA复制对小鼠卵子体外成熟、受精及发育的影响

目的 探讨胞质线粒体的氧化磷酸化（OXPHOS）活性或线粒体DNA复制在卵子成熟、受精和胚胎发育过程中的作用。方法 通过在小鼠体外成熟培养液中引入不同浓度的羰基氰4-(三氟甲氧基)苯腙 (FCCP, 10 nmol/L和100nmol/L)或2′,3′-双脱氧胞苷(ddC,10μmol/L和100μmol/L),抑制线粒体OXPHOS活性或线粒体DNA复制,统计分析各组卵子的体外生发泡破裂（GVBD）率、核成熟率、受精率及囊胚形成率,以分析线粒体功能抑制对卵子成熟、受精和胚胎发育的影响。 结果 线粒体OXPHOS活性和DNA复制功能在卵子和胚胎中所发挥的作用并不完全相同。FCCP抑制线粒体OXPHOS活性可显著降低卵子的核成熟率和囊胚形成率;但对卵子的GVBD的发生率和受精率无显著影响。而ddC抑制线粒体DNA复制不影响卵子的体外成熟和受精,但可显著抑制囊胚的形成。 结论 OXPHOS活性主要影响卵子成熟及胚胎发育;线粒体DNA复制则主要影响胚胎发育;而线粒体功能抑制不影响卵子的成熟启动和体外受精。     

Calcium at fertilization and in early development

Fertilization calcium waves are introduced, and the evidence from which we can infer general mechanisms of these waves is presented. The two main classes of hypotheses put forward to explain the generation of the fertilization calcium wave are set out, and it is concluded that initiation of the fertilization calcium wave can be most generally explained in invertebrates by a mechanism in which an activating substance enters the egg from the sperm on sperm-egg fusion, activating the egg by stimulating phospholipase C activation through a src family kinase pathway and in mammals by the diffusion of a sperm-specific phospholipase C from sperm to egg on sperm-egg fusion. The fertilization calcium wave is then set into the context of cell cycle control, and the mechanism of repetitive calcium spiking in mammalian eggs is investigated. Evidence that calcium signals control cell division in early embryos is reviewed, and it is concluded that calcium signals are essential at all three stages of cell division in early embryos. Evidence that phosphoinositide signaling pathways control the resumption of meiosis during oocyte maturation is considered. It is concluded on balance that the evidence points to a need for phosphoinositide/calcium signaling during resumption of meiosis. Changes to the calcium signaling machinery occur during meiosis to enable the production of a calcium wave in the mature oocyte when it is fertilized; evidence that the shape and structure of the endoplasmic reticulum alters dynamically during maturation and after fertilization is reviewed, and the link between ER dynamics and the cytoskeleton is discussed. There is evidence that calcium signaling plays a key part in the development of patterning in early embryos. Morphogenesis in ascidian, frog, and zebrafish embryos is briefly described to provide the developmental context in which calcium signals act. Intracellular calcium waves that may play a role in axis formation in ascidian are discussed. Evidence that the Wingless/calcium signaling pathway is a strong ventralizing signal in Xenopus, mediated by phosphoinositide signaling, is adumbrated. The central role that calcium channels play in morphogenetic movements during gastrulation and in ectodermal and mesodermal gene expression during late gastrulation is demonstrated. Experiments in zebrafish provide a strong indication that calcium signals are essential for pattern formation and organogenesis.     

Profilin and actin-related proteins regulate microfilament dynamics during early mammalian embryogenesis

BACKGROUND: Profilins are ubiquitous proteins widely distributed in animals, including humans. They regulate actin polymerization by sequestering actin monomers in association with other actin-related proteins (Arps). Actin remodelling is essential for oocyte maturation, fertilization and embryo development; yet the role of profilins in these events is not well understood. Here we investigate profilin distribution and function during bovine fertilization and early embryogenesis, and we examine profilin localization with respect to the co-distribution of other Arps. METHODS AND RESULTS: Western blotting, confocal microscopy with immunofluorescence and protein inhibition studies with antibodies were implemented. Profilin distributes inside interphase nuclei, throughout the cytoplasm and near the cell cortex at different stages of bovine oocyte maturation, fertilization and embryo development. Expression is detected through the blastocyst stage, where profilin localizes to the inner cell mass as well as trophectoderm. Profilin co-distributes with actin monomers and Arps vasodilator-stimulated phospho protein, p140mDia, Arp 3 and p80 coilin in pronucleate-stage zygotes. Antiprofilin antibodies inhibit normal embryo development by disrupting microfilaments, but not microtubules, and result in a higher concentration of profilin and p140mDia mislocalized to the cortex. CONCLUSIONS: These findings demonstrate that profilin regulates actin dynamics both within the cytoplasm and inside the nuclei of developing mammalian embryos and that its function is essential during fertilization to ensure successful development.     

Effects of griseofulvin on in vitro porcine oocyte maturation and embryo development

Griseofulvin is an orally administered antifungal drug that affects microtubule formation in vitro and interferes with microtubule dynamics in vivo as clearly shown for mitotic cells in several cell systems. This article reports the effects of griseofulvin on in vitro maturation of porcine oocytes and subsequent effects on embryo development. Our results revealed a concentration‐dependent effect on meiotic spindles with 20–40 μM griseofulvin affecting oocyte maturation, and 40 μM affecting fertilization and embryo development. These concentrations of griseofulvin did not affect mitochondrial and cortical granule distribution that also depend on microtubule and cytoskeletal functions during oocyte maturation. Specific effects on the meiotic spindle included spindle disorganization and aberrant chromosome separation displayed as prominent chromosome clusters in oocytes treated with 40 μM griseofulvin. These results strongly suggested that griseofulvin affected porcine oocyte in vitro maturation and following embryo development by disturbing microtubule dynamics. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence

It has been suggested that mitochondrial DNA defects that effect metabolic capacity may be a proximal cause of failures in oocyte maturation, fertilization, or early embryonic development. Here, the distribution of mitochondria was examined by scanning laser confocal microscopy in living human pronuclear oocytes and cleavage stage embryos, followed either by measurements of the net ATP content of individual blastomeres or anti-tubulin immunofluorescence to determine the relationship between mitochondrial distribution and microtubular organization. The results indicate that specific patterns of perinuclear mitochondrial aggregation and microtubular organization are related, and that asymmetrical mitochondrial distributions at the pronuclear stage can result in some proportion of blastomeres with reduced mitochondrial inheritance and diminished ATP generating capacity. While the inability to divide appears to be a development consequence for an affected blastomere, for the embryo, reduced competence may occur during cleavage if several blastomeres inherit a mitochondrial complement inadequate to support normal cellular functions. The findings provide a possible epigenetic explanation for the variable developmental ability expressed within cohorts of morphologically normal early cleavage stage human embryos obtained by in-vitro fertilization.     

Ü卵子体外成熟对胞质线粒体DNA拷贝和功能的影响

目的 研究卵子体外成熟（IVM）过程对胞质内线粒体的数目、功能及卵子质量的影响,进而探讨IVM卵子低发育潜能的可能机制。方法 实验小鼠随机分为IVM组和体内成熟（IVO）组;应用Real-time-PCR、免疫荧光和荧光-荧光素酶测定法,分别检测IVM和IVO来源卵子的线粒体DNA（mtDNA）拷贝数、线粒体膜电位强度、ATP含量、线粒体分布、胞质ROS水平、卵子骨架和染色体结构。 结果 相对于IVO卵子,IVM卵子的mtDNA拷贝数显著降低;而胞质ROS生成和纺锤体及染色体结构异常率则显著增加。胞质线粒体分布模型和氧化磷酸化活性在IVM和IVO卵子间差异无显著意义。结论 非生理性IVM过程显著抑制了胞质mtDNA的复制,并增加了ROS生成和纺锤体和染色体结构异常,继而可能影响卵子细胞质的成熟进程,这可部分解释IVM卵子低发育潜能的机制。