铅对小鼠卵母细胞成熟和体外受精的影响

目的：探讨铅对小鼠生殖作用的影响。方法：采用小鼠卵母细胞体外培养、体外受精的方法研究了铅对小鼠卵母细胞的成熟和体外受精的影响。结果：铅对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量,随着培养时间的延续,卵母细胞的第一极体的释放率和体外受精率都有显著提高,结论：铅可以破坏卵母细胞减数分裂进行,降低卵母细胞的受精能力,影     

卵泡刺激素和卵丘细胞对小鼠卵母细胞体外成熟的影响

目的：研究卵泡刺激素和卵丘颗粒细胞对体外培养的卵母细胞核成熟的影响。方法：未成熟卵母细胞来自人绝经期促性腺激素刺激后的小鼠卵巢。所得生殖泡期卵母细胞分成裸卵组和卵丘-卵母细胞复合物组，在分别添加0．1，0．5和1u／mL卵泡刺激素的合成人输卵管液培养液和不添加卵泡刺激素的合成人输卵管液培养液（对照组）中培养36h。培养过程中在倒置显微镜下观察各组卵母细胞生殖泡破裂和第一极体出现。出现第一极体的卵母细胞判为核成熟。结果：裸卵各卵泡刺激素组与对照组的卵母细胞生殖泡破裂发生率及第一极体排出率差别无显著性意义（P〉0．05）；卵丘-卵母细胞复合物各卵泡刺激素浓度组比对照组卵丘-卵母细胞复合物有更高的卵母细胞生殖泡破裂发生率及第一极体排出率（P〈0．05）；但各卵泡刺激素浓度组间卵丘-卵母细胞复合物的卵母细胞生殖泡破裂发生率及第一极体排出率差别无显著性意义（P〉0．05）。卵丘-卵母细胞复合物各组的卵母细胞生殖泡破裂发生率和第一极体排除率明显高于裸卵各组（P〈0．05）。结论：（1）卵丘细胞对卵母细胞的核成熟有重要作用。（2）卵泡刺激素对卵丘-卵母细胞复合物中卵母细胞核成熟有促进作用，其作用可能是通过颗粒细胞介导的。（3）卵泡刺激素对卵母细胞核成熟的促进作用可能主要是促进减数分裂的恢复。     

铅对小鼠卵母细胞成熟和体外受精的影响

目的 :探讨铅对小鼠生殖作用的影响。方法 :采用小鼠卵母细胞体外培养、体外受精的方法研究了铅对小鼠卵母细胞的成熟和体外受精的影响。结果 :铅对小鼠卵母细胞生发泡破裂没有影响 ,但可以抑制卵母细胞第一极体的释放 ,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量。随着培养时间的延续 ,卵母细胞的第一极体的释放率和体外受精率都有显著提高。结论 :铅可以破坏卵母细胞减数分裂进行 ,降低卵母细胞的受精能力 ,影响小鼠的正常生殖功能     

BDE-209对小鼠卵母细胞体外成熟的影响

目的:研究BDE-209(brominated diphenyl ethers-209)对小鼠卵母细胞体外成熟过程中第一极体释放率和卵母细胞存活率的影响.方法:通过对性成熟未孕健康母鼠进行超排卵处理,取得成熟卵母细胞,将0 μg/mL作为对照组(A组),将10、20、40μg/mL作为实验组(B组、C组、D组),用不同浓度的BDE-209进行体外培养,观察各组在8 h和16h的第一极体释放率和卵母细胞存活率,并比较其差异.结果:分别比较A、B、C、D各组8 h和16h第一极体释放率,各组差异无统计学意义(P>0.05);4组间于8 h和16 h两时段分别比较其第一极体释放率差异均无统计学意义(P>0.05);分别比较A、B、C、D各组8 h和16 h的卵母细胞存活率,A组差异无统计学意义(P>0.05),B、C、D各组16 h组比8 h组卵母细胞存活率均有所降低(P<0.05),于8、16 h时段分别比较4组间卵母细胞存活率,差异无统计学意义(P>0.05).结论:BDE-209不抑制小鼠卵母细胞第一极体释放率,但降低卵母细胞的存活率.     

细胞移植过程中体细胞克隆提高卵母细胞去核率的最佳方法

目的:治疗性克隆应用于临床中需有效解决体细胞克隆及干细胞培养两大技术。观察注射人绒毛膜促性腺激素后不同时间体内成熟卵母细胞MⅡ期染色质与第一极体相对位置,确定以第一极体为标志,盲吸法去核时间和与染色质的位置关系,解决克隆过程中卵母细胞来源与去核率。方法:实验时间为2004-09/11,实验地点为哈尔滨医科大学组织学与胚胎学教研室组织工程研究中心层流实验室。4～6周雌性昆明小鼠在明暗循环的环境饲养,做超数排卵处理。卵母细胞用含10mg/LHoechest33342的M2液染色5min,在Olymopus荧光显微镜下经紫外荧光激发,观察MⅡ期染色质与第一极体的相对位置,并且用不同的角度记录(0°～30°,30°～90°和90°～180°)。Moteic软件测量卵周隙,明确卵周隙变化规律。结果:①注射人绒毛膜促性腺激素12h,开始排卵,可见第一极体的卵母细胞占72.22%,23.08%第一极体已开始退化。排卵后,随着卵母细胞在体内的老化,退化的第一极体增多,到注射人绒毛膜促性腺激素19h退化第一极体的比率达到100%,可见第一极体的卵母细胞比率降到7.14%。②注射人绒毛膜促性腺激素12和14hMⅡ期染色质与第一极体位置比邻,在0°～30°区域的卵母细胞较其他时间段多,为61.54%和50%;注射人绒毛膜促性腺激素13和17h的卵母细胞MⅡ期染色质与第一极体位置以在30°～90°区域为主;注射人绒毛膜促性腺激素15hMⅡ期染色质与第一极体位置在0°～30°和90°～180°区域的卵母细胞数相等,为30.77%,且少于30°～90°区域的卵母细胞(38.46%);注射人绒毛膜促性腺激素16,18和19hMⅡ期染色质与第一极体位置在90°～180°区域的卵母细胞逐渐增多,注射人绒毛膜促性腺激素19h达到最大值66.67%。③卵周隙随卵母细胞在体内的老化逐渐增大。结论:注射人绒毛膜促性腺激素12h可以进行小鼠卵母细胞去核操作,注射人绒毛膜促性腺激素14h为最佳去核时间。     

细胞移植过程中体细胞克隆提高卵母细胞去核率的最佳方法

目的：治疗性克隆应用于临床中需有效解决体细胞克隆及干细胞培养两大技术。观察注射人绒毛膜促性腺激素后不同时间体内成熟卵母细胞MⅡ期染色质与第一极体相对位置，确定以第一极体为标志，盲吸法去核时间和与染色质的位置关系，解决克隆过程中卵母细胞来源与去核率。方法：实验时间为2004-09／11，实验地点为哈尔滨医科大学组织学与胚胎学教研室组织工程研究中心层流实验室。4～6周雌性昆明小鼠在明暗循环的环境饲养，做超数排卵处理。卵母细胞用含10mg／L Hoechest33342的M2液染色5min，在Olymopus荧光显微镜下经紫外荧光激发，观察MⅡ期染色质与第一极体的相对位置，并且用不同的角度记录(0&;#176;～30&;#176;，30&;#176;～90&;#176;和90&;#176;～180&;#176;)。Moteic软件测量卵周隙，明确卵周隙变化规律。结果：①注射人绒毛膜促性腺激素12h，开始排卵，可见第一极体的卵母细胞占72．22％，23．08％第一极体已开始退化。排卵后，随着卵母细胞在体内的老化，退化的第一极体增多，到注射人绒毛膜促性腺激素19h退化第一极体的比率达到100％，可见第一极体的卵母细胞比率降到7．14％。②注射人绒毛膜促性腺激素12和14h MⅡ期染色质与第一极体位置比邻，在0&;#176;～30&;#176;区域的卵母细胞较其他时间段多，为61．54％和50％；注射人绒毛膜促性腺激素13和17h的卵母细胞MⅡ期染色质与第一极体位置以在30&;#176;～90&;#176;区域为主；注射人绒毛膜促性腺激素15hMⅡ期染色质与第一极体位置在0&;#176;～30&;#176;和90&;#176;～180&;#176;区域的卵母细胞数相等，为30．77％，且少于30&;#176;～90&;#176;区域的卵母细胞(38．46％)；注射人绒毛膜促性腺激素16，18和19hMⅡ期染色质与第一极体位置在90&;#176;～180&;#176;区域的卵母细胞逐渐增多，注射人绒毛膜促性腺激素19h达到最大值66．67％。③卵周隙随卵母细胞在体内的老化逐渐增大。结论：注射人绒毛膜促性腺激素12h可以进行小鼠卵母细胞去核操作，注射人绒毛膜促性腺激素14h为最佳去核时间。     

近交系小鼠体细胞核移植胚胎的构建和鉴定

背景：体细胞核移植技术己成功用于多种动物的克隆，但其过程中核移植技术的成功率较低。 目的：通过对卵母细胞不同去核方法的对比分析，观察其对小鼠体细胞核移植胚胎体外发育的影响，并利用微卫星DNA技术进行核移植囊胚鉴定，幸刀步建立小鼠体细胞核移植技术平台。 设计、时间及地点：以卵母细胞为观察对象的对比实验，于2005—09/2007-10在广西医科大学医学科学实验中心及动物实验中心完成。材料：选用C57BL／6小鼠卵母细胞为受体、BALB／c小鼠卵丘细胞为供体。取600枚受体卵母细胞，根据去核方法不同分为3组。每组200枚。 方法：①盲吸法：吸出卵母细胞第1极体及其附近的适量胞质。②蔗糖辅助去核法：以含蔗糖显微操作液预处理卵母细胞，吸出可见的细胞核等遗传物质。③荧光染色去核法：Hoechest33342预处理卵母细胞，在紫外光下用去除遗传物质。乙醇联合二甲氨基嘌呤进行重构胚激活，激活后卵裂培养基液滴中培养。根据Mouse Genome Database设计小鼠微卫星DNA多态聚合酶链反应引物，提取近交系小鼠体细胞核移植囊胚、供体BALB／c小鼠、受体C57BL／6小鼠及昆明小鼠的基因组DNA，使用巢式聚合酶链反应扩增选定的序列。最终扩增出4个微卫星位点DNA片段，即D3Mit28，D11Mit258，D12Mit136及D14Mit50。主要观察指标：比较3种去核方法的去核率、卵裂率、囊胚率及囊胚细胞数差异。对巢式聚合酶链反应产物进行琼脂糖凝胶电泳验证。 结果：①盲吸去孩法的去核率、处理重构胚的体外发育能力均低于荧光染色去核法和蔗糖辅助去核法（P〈0.05）。②荧光染包去核法的去核率高于蔗糖辅助去核法（P〈0.05），两组的囊胚率及囊胚细胞数差异均无显著性意义（P〉0.05）。③巢式聚合酶链反应可扩增微量基因组DNA。通过微卫星DNA序列的扩增，证明核移植囊胚的微卫星DNA与供体细胞完全相同，而与受体细胞或者对照细胞无亲缘关系。 结论：荧光染色去核法和蔗糖辅助去核法适用于小鼠核移植，可支持小鼠体细胞核移植胚胎的早期发育。微卫星分析证实体细胞核移植囊胚为供体BALB/c小鼠的克隆，微卫星DNA分析可用于小鼠体细胞核移植囊胚的鉴定。     

Hela细胞MPF对未成熟卵母细胞的体外促成熟作用分析

【目的】研究Hela细胞M期促进因子MPF对未成熟卵母细胞的体外促成熟作用的生物活性,并观察单个成熟卵母细胞的中期染色体。【方法】应用秋水仙胺将Hela细胞阻滞在有丝分裂的中期（meta—phase）,从中提取MPF蛋白;体外培养基分为无激素组、有激素素组、注射组,将MPF提取物通过显微注射,分别导入3组培养基中的昆明小白鼠未成熟卵母细胞中,观察未成熟卵母细胞发生GVBD（生发泡破裂）、或者排出极体的数量,并对3组两两之间发生GVBD或者排出极体的情况,作统计分析;以及制备并观察成熟卵母细胞的中期相染色体。【结果】经过统计分析说明,发生GVBDD的情况,在注射组与无激素组之间差异有显著性（P〈0．05）;排出极体的情况,在注射组与无激素组之间,以及注射组与激素组之间差异有显著（P〈0．005,P〈0．05）。已经观察到了部分成熟卵母细胞的中期染色体。【结论】MPF提取物具有诱导小白鼠未成熟卵母细胞在体外提前恢复减数分裂的生物活性。     

人类不成熟卵体外培养的应用

1935年Pincus和Enzmann在家兔的实验中发现,若将不成熟卵母细胞从卵泡中释放出来,它在体外适当培养基中可发育成熟.这一现象使人们开始联想到从体内抽取的不成熟卵母细胞可在体外培养成熟,并可用来挽救发育中即将退化的卵母细胞,同时可能应用于临床提供卵子用于体外受精.人不成熟卵细胞的体外成熟(in vitro maturation, IVM),由Cha[1]首先报道成功从手术穿刺卵巢获得不同成熟阶段的未成熟卵母细胞,在体外培养到成熟卵母细胞而进行体外受精和胚胎移植.随后Trounson等[2]报道在阴道B超引导下穿刺2～10 mm直径的卵泡,未接受促激素刺激的多囊卵巢综合征(PCOS)患者卵巢获得不成熟卵进行体外培养并获得成功妊娠.     

自然周期 IVF／M 手术管理及护理对策

目的：探讨自然周期IVF/M手术的管理模式。方法通过建立规章制度,人员管理、患者管理制度,做好患者的手术管理、健康管理,减轻患者术前心理焦虑及紧张状态,达到获得成熟卵母细胞和未成熟卵母细胞为目的。结果专科特殊性迫切需要建立一套完整的管理模式。结论根据自然周期IVF/M手术管理的特点,实施全程人性化服务,进行心理干预,是促进自然周期IVF/M技术成功应用的保障,是保证成熟卵母细胞和未成熟卵母细胞获得的关键,同时也是体现自然周期IVF/M手术管理的关键。     

Meiotic Regulation of TPX2 Protein Levels Governs Cell Cycle Progression in Mouse Oocytes

Formation of female gametes requires acentriolar spindle assembly during meiosis. Mitotic spindles organize from centrosomes and via local activation of the RanGTPase on chromosomes. Vertebrate oocytes present a RanGTP gradient centred on chromatin at all stages of meiotic maturation. However, this gradient is dispensable for assembly of the first meiotic spindle. To understand this meiosis I peculiarity, we studied TPX2, a Ran target, in mouse oocytes. Strikingly, TPX2 activity is controlled at the protein level through its accumulation from meiosis I to II. By RNAi depletion and live imaging, we show that TPX2 is required for spindle assembly via two distinct functions. It controls microtubule assembly and spindle pole integrity via the phosphorylation of TACC3, a regulator of MTOCs activity. We show that meiotic spindle formation in vivo depends on the regulation of at least a target of Ran, TPX2, rather than on the regulation of the RanGTP gradient itself.     

Small GTPases and formins in mammalian oocyte maturation: cytoskeletal organizers

The maturation process of mammalian oocytes accompanies an extensive rearrangement of the cytoskeleton and associated proteins. As this process requires a delicate interplay between the cytoskeleton and its regulators, it is often targeted by various external and internal adversaries that affect the congression and/or segregation of chromosomes. Asymmetric cell division in oocytes also requires specific regulators of the cytoskeleton, including formin-2 and small GTPases. Recent literature providing clues regarding how actin filaments and microtubules interact during spindle migration in mouse oocytes are highlighted in this review.     

Cyclic nucleotide phosphodiesterase 3A-deficient mice as a model of female infertility

Since cAMP blocks meiotic maturation of mammalian and amphibian oocytes in vitro and cyclic nucleotide phosphodiesterase 3A (PDE3A) is primarily responsible for oocyte cAMP hydrolysis, we generated PDE3A-deficient mice by homologous recombination. The Pde3a(-/-) females were viable and ovulated a normal number of oocytes but were completely infertile, because ovulated oocytes were arrested at the germinal vesicle stage and, therefore, could not be fertilized. Pde3a(-/-) oocytes lacked cAMP-specific PDE activity, contained increased cAMP levels, and failed to undergo spontaneous maturation in vitro (up to 48 hours). Meiotic maturation in Pde3a(-/-) oocytes was restored by inhibiting protein kinase A (PKA) with adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) or by injection of protein kinase inhibitor peptide (PKI) or mRNA coding for phosphatase CDC25, which confirms that increased cAMP-PKA signaling is responsible for the meiotic blockade. Pde3a(-/-) oocytes that underwent germinal vesicle breakdown showed activation of MPF and MAPK, completed the first meiotic division extruding a polar body, and became competent for fertilization by spermatozoa. We believe that these findings provide the first genetic evidence indicating that resumption of meiosis in vivo and in vitro requires PDE3A activity. Pde3a(-/-) mice represent an in vivo model where meiotic maturation and ovulation are dissociated, which underscores inhibition of oocyte maturation as a potential strategy for contraception.     

Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics

The real-time dynamics of the T cell receptor (TCR) reflect antigen detection and T cell signaling, providing valuable insight into the evolving events of the immune response. Despite considerable advances in studying TCR dynamics in simplified systems in vitro, live imaging of subcellular signaling complexes expressed at physiological densities in intact tissues has been challenging. In this study, we generated a transgenic mouse with a TCR fused to green fluorescent protein to provide insight into the early signaling events of the immune response. To enable imaging of TCR dynamics in naive T cells in the lymph node, we enhanced signal detection of the fluorescent TCR fusion protein and used volumetric masking with a second fluorophore to mark the T cells expressing the fluorescent TCR. These in vivo analyses and parallel experiments in vitro show minimal and transient incorporation of TCRs into a stable central supramolecular activating cluster (cSMAC) structure but strong evidence for rapid, antigen-dependent TCR internalization that was not contingent on T cell motility arrest or cSMAC formation. Short-lived antigen-independent TCR clustering was also occasionally observed. These in vivo observations demonstrate that varied TCR trafficking and cell arrest dynamics occur during early T cell activation.     

High-resolution three-dimensional in vivo imaging of mouse oviduct using optical coherence tomography

The understanding of the reproductive events and the molecular mechanisms regulating fertility and infertility in humans relies heavily on the analysis of the corresponding phenotypes in mouse models. While molecular genetic approaches provide significant insight into the molecular regulation of these processes, the lack of live imaging methods that allow for detailed visualization of the mouse reproductive organs limits our investigations of dynamic events taking place during the ovulation, the fertilization and the pre-implantation stages of embryonic development. Here we introduce an in vivo three-dimensional imaging approach for visualizing the mouse oviduct and reproductive events with micro-scale spatial resolution using optical coherence tomography (OCT). This method relies on the natural tissue optical contrast and does not require the application of any contrast agents. For the first time, we present live high-resolution images of the internal structural features of the oviduct, as well as other reproductive organs and the oocytes surrounded by cumulus cells. These results provide the basis for a wide range of live dynamic studies focused on understanding fertility and infertility.OCIS codes: (110.4500) Optical coherence tomography, (170.3880) Medical and biological imaging, (170.5380) Physiology     

Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.     

PhaseRMiC: phase real-time microscope camera for live cell imaging

We design a novel phase real-time microscope camera (PhaseRMiC) for live cell phase imaging. PhaseRMiC has a simple and cost-effective configuration only consisting of a beam splitter and a board-level camera with two CMOS imaging chips. Moreover, integrated with 3-D printed structures, PhaseRMiC has a compact size of 136×91×60 mm³, comparable to many commercial microscope cameras, and can be directly connected to the microscope side port. Additionally, PhaseRMiC can be well adopted in real-time phase imaging proved with satisfied accuracy, good stability and large field of view. Considering its compact and cost-effective device design as well as real-time phase imaging capability, PhaseRMiC is a preferred solution for live cell imaging.     

实时网络平台实景的康复治疗学教学的效果

目的：研究评价基于QQ实时网络平台实景教学的康复治疗学教学效果。方法：将南京中医药大学康复治疗学专业67名学生,随机分为课堂教学组（课堂组）34名和QQ网络平台实景教学组（网络组）33名,分别采用普通课堂教学法与QQ实时网络平台实景教学法。2学时教学后采用教育环境测量（DREEM）表调查问卷对教学效果进行评价。结果：课堂组和网络组分别收回31份（91.2%）、29份（87.9%）有效问卷。DREEM显示2组总分均较高,但网络组明显高于课堂组。各单项目分中,网络组在学习、教师、环境及学术自我知觉以及总分5方面评分均明显高于课堂组（P〈0.05）。结论：实时网络平台实景教学可提高学生学习动机,改善教育环境和教学质量,是一种新型、有效、便捷、低成本的教学模式。     

In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse

Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.OCIS codes: (170.2655) Functional monitoring and imaging, (170.3010) Image reconstruction techniques, (170.3650) Lifetime-based sensing, (170.3660) Light propagation in tissues, (170.3880) Medical and biological imaging, (170.6960) Tomography