不同发育阶段斑马鱼胚胎对X射线辐射敏感性差异

目的:考察X射线对斑马鱼早期胚胎发育的影响,探讨其影响机制.方法:用X射线照射不同发育时段的斑马鱼胚胎,统计死亡率和畸形率;应用单细胞凝胶电泳(SCGE)技术检测X射线对胚胎DNA损伤的影响;采用DCFH-DA测定胚胎活性氧;总抗氧化能力(T-AOC)测定;结果:X射线照射对斑马鱼胚胎发育有明显的影响,能够导致胚胎发育畸形如围心腔水肿、脊柱扭曲、尾部弯曲等多种畸形甚至死亡;检测X射线对斑马鱼胚胎DNA损伤时,发现X射线照射对胚胎中的DNA能够产生明显的损伤,且DNA损伤程度随胚胎发育的进行而减弱;胚胎经X射线照射后活性氧的产生增加;胚胎总抗氧化能力随着胚胎发育的进行而逐渐增强;结论:X射线照射明显影响斑马鱼胚胎发育,并造成胚胎细胞DNA损伤,发育早期胚胎敏感性高;发育后期胚胎对X射线敏感性降低,可能与胚胎细胞抗氧化能力增强有关.     

小鼠胚胎体外培养条件的研究

为了探讨小鼠胚胎在体外发育过程中的最佳方案,我们将从超排昆明小鼠取出的５４３枚受精卵经过不同的培养液（Ｍ１６、Ｍ１６＋ＯＥＣ、Ｇｌｕ－ＦｒｅｅＭ１６和Ｇｌｕ－ＦｒｅｅＭ１６＋ＯＥＣ）及不同的培养微环境处理后,观察胚胎体外发育的过程。结果表明,葡萄糖在胚胎发育早期（２细胞期）有比较明显的发育阻断作用,同种输卵管上皮（ＯＥＣ）共同培养能够有效地抑制这种阻断作用;葡萄糖对胚胎８－１６细胞期及以后阶段的发育具有十分重要的作用,而此时ＯＥＣ的作用则不明显;除了葡萄糖外,在早期可能还有其它一些象磷酸盐、重金属离子的物质也能起胚胎发育阻断作用。同时还发现培养微环境的稳定也是胚胎发育的重要保证条件,石蜡油的封盖能够保持某种特定微环境而支持胚胎的体外发育。     

葡萄糖对小鼠胚胎体外发育的影响

通过在CZB培养液中添加不同浓度葡萄糖及在胚胎发育的不同阶段加入葡萄糖,对小鼠胚胎进行体外培养,以探讨葡萄糖在小鼠早期胚胎体外发育中的作用。其结果表明,小鼠胚胎在含糖CZB与在无糖CZB中培养比较,4-细胞发育率无差异;各浓度葡萄糖组囊胚率显著高于无糖组,其中3.0mmol/L浓度组囊胚细胞数显著高于其余组;实验二：2-细胞至4-细胞、4-细胞至桑椹胚前添加葡萄糖囊胚率显著提高。上述结果证明,在小鼠胚胎体外培养中加入葡萄糖不会导致2-细胞阻滞;葡萄糖浓度增加至10mmol/L对小鼠胚胎无毒性作用,其最适浓度为3.0mmol/L;2-细胞至4-细胞、4-细胞至桑椹胚前添加葡萄糖是必要的。关键词 葡萄糖;小鼠;2-细胞阻滞;胚胎;体外发育     

低温和变温诱导对飞蝗胚胎发育的影响

【目的】飞蝗Locusta migratoria(Linnaeus)是我国乃至全世界广泛发生的重要农业害虫,其种群暴发会给农作物造成重大为害和减产。飞蝗种群发生动态受低温及变温的影响,但低温和变温如何作用于胚胎发育和是否诱导胚胎滞育的发生,目前尚不清楚。【方法】本文在对飞蝗胚胎发育特点观察鉴定的基础上,研究了胚胎发育中期恒定低温诱导、诱导后恢复、发育中期变温诱导及发育早期低温诱导等条件对胚胎发育进程的影响。【结果】研究发现,胚胎发育起点温度下非致死温度7.5℃处理促使胚胎发育历期变异增加,而25~30℃高温下胚胎发育整齐。7.5℃处理时间长短对胚胎发育影响不明显,但该低温培育时间长短影响后期常温下的胚胎发育,即7.5℃下长期低温可能促进25℃或30℃下的胚胎的发育。25℃以下变温温度影响胚胎发育进展,但影响胚胎发育的限制因子主要是发育起点以上温度。【结论】因此,温度作为单一环境因子,其特定的低温和变温处理不仅没有诱导胚胎滞育的发生,反而促进后期胚胎在常温下的发育。本研究结果对揭示温度变化对个体发育和种群动态影响及预测飞蝗种群发生有重要的指导意义。     

单个植入前胚胎构建cDNA文库

以PCR为基础的单个植入前胚胎cDNA文库构建是一种快速、可重复和高效的建库方法。单个植入前胚胎cDNA文库作为一种重要的新兴基因资源库 ,为新基因的克隆和鉴定奠定了坚实的基础 ,不仅解决了胚胎研究材料受限的问题 ,而且在时间上更加精确 ,更符合胚胎发育的规律。是研究早期胚胎发育基因表达的一种有效手段。随着这一技术的不断改进和与其它技术的有机结合 ,必然为人们进一步揭示胚胎发育的分子机制开创一个崭新的局面。     

葡萄糖对胚胎发育的致畸作用研究

研究发现妊娠糖尿病是影响胚胎发育的重要致畸因素之一,患糖尿病孕妇的胎儿发生先天畸形风险性较非糖尿病孕妇胎儿高3-5倍,糖尿病妊娠导致心血管系统、神经系统的先天畸形严重影响胎儿及新生儿的生存质量。本实验分离孕9．5d的SD大鼠胚胎,采用体外全胚培养技术研究葡萄糖对胚胎发育的影响。实验分组：1．正常对照组,     

高浓度葡萄糖对昆明小鼠早期胚胎发育的影响

建立昆明小鼠受孕模型,分离并体外培养胚胎细胞.检测了各培养浓度下的细胞增殖、分化与凋亡.胚胎细胞在0.2mmol/L和5.56mmol/L葡萄糖浓度的KSOM培养基中能正常发育和孵化;而在浓度为15.56mmol/L和25.56mmol/L葡萄糖培养基中胚胎发育和孵化均受到损害(P<0.005),且总细胞数和内细胞团细胞数也明显减少(P<0.01),但其细胞凋亡率与0.2mmol/L和5.56mmol/L葡萄糖浓度下胚胎细胞凋亡率无显著性差异(P>0.05).随着葡萄糖浓度的增高,胚泡总的表面积无明显变化,但胚胎细胞密度呈增加趋势.高血糖对早期胚胎的发育具有毒性作用,提示高糖可能导致妊娠合并糖尿病患者的流产和胎儿畸形率升高.     

迷迭香酸对氯化镉致斑马鱼胚胎发育毒性的保护作用北大核心CSCD

通过考察氯化镉(CdCl2)对斑马鱼胚胎发育的毒性效应及迷迭香酸(rosmarinic acid,RA)的干预作用,探讨镉的发育毒性机理。将受精1 h后的斑马鱼胚胎暴露于不同浓度的CdCl2或含不同浓度RA的CdCl2溶液中,观察胚胎死亡、孵化及幼鱼畸形的情况。采用吖啶橙染色,定性观察胚胎细胞凋亡情况;以单细胞凝胶电泳法检测胚胎细胞的DNA损伤;以活性氧(reactive oxygen species,ROS)荧光探针DCFH-DA染色法检测胚胎的ROS水平,硫代巴比土酸比色法测定胚胎脂质过氧化水平,5,5-二硫二硝基苯甲酸比色法测定胚胎的还原谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)水平;光泽精化学发光法检测胚胎还原型辅酶(nicotinamide adenine dinucleotide phosphate,NADPH)Ⅱ氧化酶(NADPH oxidase,Nox)活性。结果显示,RA浓度依赖性地抑制了CdCl2诱导的胚胎死亡和幼鱼畸型,同时提高了胚胎孵化率。RA可浓度依赖性地改善CdCl2诱导的胚胎ROS和MDA(malonic dialdehyde)水平升高、GSH/GSSG比值降低和Nox活性升高,抑制CdCl2诱导的胚胎细胞凋亡。RT-PCR结果显示,RA对CdCl2诱导的胚胎Cu/Zn-Sod基因表达上调和Bcl-2/Bax水平下调也有明显改善作用。结果说明,CdCl2对斑马鱼胚胎发育具有毒性,可活化Nox,促进ROS生成,造成胚胎细胞氧化胁迫损伤以至胚胎细胞凋亡,而RA可抑制Nox活化,改善氧化胁迫状态,从而对CdCl2的发育毒性起保护作用。     

鼠单性生殖获得成功

据英国《新科学家》杂志最近报道,可以不需要来自雄性的遗传物质,鼠的胚胎也会发育成熟。这一迄今尚未发表的令人惊奇的成果,来自两位在遗传学研究最前沿的杰出的科学家,它使当代胚胎发育的理论产生混乱,并将导致关于精子对胚胎生存所起作用以及胚胎发育的更深刻的了解。     

小鼠母源因子对早期胚胎发育的影响

在脊椎动物中发育过程中,卵母细胞要经历MII期停滞、受精、早期胚胎发育的启动、胚胎基因组的转录激活、并指导完成个体的发育过程。同时,核移植过程中,分化的细胞核在去核的卵母细胞中能够重编程到胚胎早期的状态并能完成个体的发育过程。在这些发育过程中母源因子都发挥了极其的重要作用。在小鼠胚胎发育研究中发现,小鼠的基因组激活发生在2细胞期,这一时期标志着合子的发育由卵母细胞控制向胚胎控制的过渡,期间发生一系列复杂的生化过程。体外培养的小鼠的胚胎的发育阻断也易发生的2细胞时期。因此对卵母细胞及早期胚胎母源因子的研究,将有利于了解早期体外培养胚胎和克隆胚胎发育失败的原因,为提高体外培养和克隆胚胎发育的成功率提供理论的基础。     

Methionine and iron as growth factors for rat embryos cultured in canine serum

Development of headfold-staged rat embryos cultured in canine serum containing various supplements was compared with development in rat serum to seek suitable alternatives to rat serum in rodent embryo culture and to identify nutritional factors for cultured rodent embryos that may have relevance for normal mammalian embryonic growth and development. Supplementation of canine serum with glucose, methionine, and a lipophilic iron chelate allowed growth and development of cultured rat embryos, approximating those obtained with rat serum. These findings suggest that properly supplemented canine serum can serve as a suitable rodent embryo culture medium and that glucose, iron, and methionine may be important nutrients in mammalian embryonic development.     

In vitro analysis of glucose metabolism and embryonic growth in postimplantation rat embryos

The glucose metabolism and embryonic development of rat embryos during organogenesis was studied using embryo culture. Glucose uptake and embryonic growth and differentiation of 10.5-day explants (embryos + membranes) were limited by the decreasing glucose concentration, but not the increasing concentration of metabolites, in the culture media during the second 24 h of a 48 h culture. No such limitations were found on the embryonic development of 9.5-day explants during a 48 h culture although glucose uptake was slightly reduced at very low concentrations of glucose. From the head-fold stage to the 25-somite stage of development, glucose uptake was characteristic of the stage of development of the embryo and not the time it had been in culture. Embryonic growth of 9.5-day explants was similar to that previously observed in vivo. Glucose uptake by 9.5-day explants was dependent on the surface area of the yolk sac and was independent of the glucose concentration in the culture media (within the range of 9.4 to 2.5 mM). The proportion of glucose converted to lactate was 100% during the first 42h of culture then fell to about 50% during the final 6h. The protein contents of both the extraembryonic membranes and the embryo were dependent on the glucose uptake.     

Glucose transporter isoform-3 mutations cause early pregnancy loss and fetal growth restriction

Glucose transporter isoform-3 (GLUT3) is the trophoblastic facilitative glucose transporter. To investigate the role of this isoform in embryonic development, we created a novel GLUT3-null mouse and observed arrested early embryonic development and loss at neurulation stage when both alleles were mutated. This loss occurred despite the presence of other related isoforms, particularly GLUT1. In contrast, when a single allele was mutated, despite increased embryonic cell apoptosis, adaptive changes in the subcellular localization of GLUT3 and GLUT1 in the preimplantation embryo led to postimplantation survival. This survival was compromised by decreased GLUT3-mediated transplacental glucose transport, causing late-gestation fetal growth restriction. This yielded young male and female adults demonstrating catch-up growth, with normal basal glucose, insulin, insulin-like growth factor-I and IGF-binding protein-3 concentrations, fat and lean mass, and glucose and insulin tolerance. We conclude that GLUT3 mutations cause a gene dose-dependent early pregnancy loss or late-gestation fetal growth restriction despite the presence of embryonic and placental GLUT1 and a compensatory increase in system A amino acid placental transport. This critical life-sustaining functional role for GLUT3 in embryonic development provides the basis for investigating the existence of human GLUT3 mutations with similar consequences during early pregnancy.     

Requirement for glucose during in vitro culture of sheep preimplantation embryos.

Glucose utilization by sheep embryos was examined in 8-cell (N = 36) and blastocyst (N = 36) stages, by measuring conversion of [5-3H]glucose to 3H2O. Fifty percent glucose utilization occurred at 0.79 +/- 0.69 mM for 8-cell embryos and -0.06 +/- 0.15 mM for blastocysts. Development of 1- and 2-cell sheep embryos (N = 264) was examined under different glucose concentrations (0, 1.5, 3, or 6 mM) and in the presence or absence of 0.33 mM pyruvate and 3.3 mM lactate (PL). Overall, the presence of glucose was detrimental (P less than 0.001) to embryonic development. By contrast, the presence of pyruvate and lactate was beneficial (P less than 0.001) to development. An interaction was observed between the concentration of glucose and presence or absence of PL (P less than 0.05). An optimum level of glucose occurs at 0-3 mM in the presence of PL (P less than 0.1). Development to the blastocyst stage was observed in medium when supplemented with amino acids and albumin alone. Thus, glucose metabolism is not critical for embryonic development, but beneficial at low concentrations. High concentrations can inhibit development, possibly by inhibiting the tricarboxylic acid (TCA) cycle. Sheep embryos may also be using amino acids as an energy source for development.     

GLUT1 deficiency links nutrient availability and apoptosis during embryonic development

GLUT1 is essential for human brain development and function, as evidenced by the severe epileptic encephalopathy observed in children with GLUT1 deficiency syndrome resulting from inherited loss-of-function mutations in the gene encoding this facilitative glucose transporter. To further elucidate the pathophysiology of this disorder, the zebrafish orthologue of human GLUT1 was identified, and expression of this gene was abrogated during early embryonic development, resulting in a phenotype of aberrant brain organogenesis consistent with the observed expression of Glut1 in the embryonic tectum and specifically rescued by human GLUT1 mRNA. Affected embryos displayed impaired glucose uptake concomitant with increased neural cell apoptosis and subsequent ventricle enlargement, trigeminal ganglion cell loss, and abnormal hindbrain architecture. Strikingly, inhibiting expression of the zebrafish orthologue of the proapoptotic protein Bad resulted in complete rescue of this phenotype, and this occurred even in the absence of restoration of apparent glucose uptake. Taken together, these studies describe a tractable system for elucidating the cellular and molecular mechanisms of Glut1 deficiency and provide compelling in vivo genetic evidence directly linking nutrient availability and activation of mitochondria-dependent apoptotic mechanisms during embryonic brain development.     

Carbohydrate metabolism during embryonic and larval development of Odonthestes humensis(De Buen, 1953) (Pisces—Atherinidae)

Changes in glucose and glycogen concentrations during embryonic and larval development of Odonthestes humensis (De Buen, 1953) (Pisces—Atherinidae) were followed.
Glycogen decreased immediately after fertilization but remained constant throughout the embryonic period, suggesting that although present glycogenolisis may not be the most important energetic pathway during this period. Glucose levels only show a significant increase on the fifth day of development coincident with the beginning of heart activity.
In fed larvae glycogen utilization increases during development, resulting in an increase in glucose concentration. Such a pattern does not occur in starved animals, where glycogen and glucose are present respectively at high and low concentrations similar to embryos. The change of pattern seems to be associated with the first larval feeding.     

Expressional changes of ganglioside GM3 during ovarian maturation and early embryonic development in db/db mice

Diabetes and obesity cause abnormal development of reproductive processes in a variety of species, but the mechanisms that underlie this effect have not been fully elucidated. This study examined the expressional changes of ganglioside GM3 during ovarian maturation, in vitro fertilization (IVF) and early embryonic development in diabetic/obese db/db mice. In high-performance thin-layer chromatography studies, GM3 expression was conspicuously low in the ovaries of db/db mice compared to non-diabetic db/+ mice. Signal detected by anti-GM3 monoclonal antibody was greatly reduced in the primary, secondary and graffian follicles of db/db mice compared to control mice. Results from IVF with ova and sperm from db/db mice showed that GM3 expression during early embryonic development was obviously decreased compared to db/+ mice. This study also elucidated the effects of high glucose (20 and 30 mm) on early embryonic development in ICR strain mice. High glucose caused a decrease in GM3 expression during early embryonic development. Taken together, the results of this study indicate decreased GM3 expression during ovarian maturation and embryonic development of db/db mice, suggesting that alteration of ganglioside expression induced by the diabetic condition may be implicated in the abnormal follicular embryonic development.     

Developmental regulation in the expression of rat heart glucose transporters.

Antibodies against human erythrocyte glucose transporters (GLUT-1) were used to determine if the transporters of embryonic and adult rat hearts have similar reactivity. On the basis of immunoblotting, these antibodies react more strongly with embryonic transporters than with adult ones. To determine if this phenomenon may be correlated with changes in the expression of transporter types during development, RNA isolated from either the embryonic or the adult rat heart was amplified by polymerase chain reaction (PCR) to identify the transporter species. Both GLUT-1 and GLUT-4 fragments were obtained among the PCR products. They were used for Northern blot analysis. The results indicate that the embryonic heart is rich in GLUT-1 mRNA; whereas the adult heart contains predominantly GLUT-4 mRNA. Thus, it appears that the major type of glucose transporter in rat heart switches from GLUT-1 to GLUT-4 during development.     

GLYCOGEN METABOLISM AND CHANGES IN THE ACTIVITIES OF PHOSPHORYLASE, PHOSPHOFRUCTOKINASE AND PYRUVATE KINASE DURING DEVELOPMENT OF SEA URCHIN EGGS

The glucose and glycogen contents of sea urchin eggs and embryos were measured enzymatically. Unfertilized eggs of Hemicentrotus pulcherrimus and Anthocidaris crassispina contain about 20.9 and 24.4 μg of glycogen per mg protein, respectively. As for glucose, unfertilized eggs of Hemicentrotus and Anthocidaris contain about 0.7 and 1.9 μg per mg protein, respectively. Glycogen consumption during embryonic development differs with different species of sea urchins. In Anthocidaris , glycogen decreases significantly after fertilization. The oxidation of glucose and glycogen accounts for about 50% of oxygen consumed until the early blastula stage in this species. The contribution ratio of glucose and glycogen to the overall energy pool becomes less than 10% at later stages. In Hemicentrotus , however, the glycogen content remains unchanged until the early blastula stage and thereafter decreases. The importance of glucose and glycogen as an energy fuel seems little throughout the development of Hemicentrotus. Activities of phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) were measured at various embryonic stages in both species of sea urchins. The difference between two species in the consumption of glucose and glycogen can not be elucidated by the differences in the activities of these enzymes.