UVB影响中华大蟾蜍早期胚胎发育的初步研究

为了研究UVB对两栖类动物早期胚胎发育的影响,以中华大蟾蜍为研究对象,用UVB分别以不同强度和不同时间照射其二细胞期胚胎,观察记录胚胎的发育过程、统计死亡率并在形态学上对UVB照射诱导的畸胚进行分析。结果表明,30μW／cm^2强度的UVB分别照射二细胞期胚胎20s、60s和80μW／cm。强度分别照射20s、60s、120s后,对中华大蟾蜍早期胚胎发育有促进作用,使发育过程中的死亡率降低,畸胚出现的类型较少且畸变程度低;80μW／cm。强度的UVB照射二细胞期胚胎600s,100μW／cm^2强度的UVB照射20s、60s、600s,120μw／cm。强度的UVB照射20s,导致胚胎发育过程中死亡率明显升高,畸胚数量多且畸变程度高。结论：UVB照射剂量和时间不同,对中华大蟾蜍早期胚胎发育的影响有明显差异。     

激光照射对中华大蟾蜍原肠胚蛋白质表达的影响

用YAG倍频激光器选择不同能量密度对中华大蟾蜍原肠胚进行照射,对各组样品进行双向电泳,凝胶成像后用PDQuest软件进行分析,结果表明:激光照射对蛋白质点数及其分布影响显著,影响程度与照射激光密度有关。各激光照射组与对照组相比,有些蛋白质点变化明显,有些蛋白质点在对照组未发现,而在各激光照射组皆新出现。结论:激光照射可对原肠胚一些蛋白质的表达产生明显的影响。     

激光照射对中华大蟾蜍早期胚胎染色体的影响

应用YAG倍频激光器对中华大蟾蜍四细胞期胚胎进行照射,观察胚胎的生长、发育,并对激光照射致畸的胚胎在尾芽期用压片法进行染色体分析。实验结果表明：激光照射后正常的胚胎其染色体无变化,激光照射致畸的胚胎其染色体有丢失和加倍等变化。     

乙草胺对中华大蟾蜍(Bu fo bu fo gar garizans)早期胚胎发育的影响

用不同浓度的乙草胺分别对中华大蟾蜍 4个不同发育时期的早期胚胎作用 5小时 ,结果发现 ,不同浓度的乙草胺可以导致中华大蟾蜍早期胚胎不同程度的发育成原肠外突和畸胎。不同发育时期的中华大蟾蜍早期胚胎对乙草胺的敏感程度和耐受程度都有所不同 ,其中 ,原肠胚期的胚胎对乙草胺最为敏感。实验表明乙草胺对中华大蟾蜍早期胚胎发育具有明显的致畸致死效应。     

He—Ne激光辐照天使鱼胚胎导致畸变

采用He—Ne激光(632.8nm辐照了天使鱼(Pterophyllum eimeki)的发育阶段的胚胎,观察和分析激光辐照对胚胎发育的致变影响和各发育阶段胚胎对激光的受激反应。对受精卵、囊胚期、原肠期和神经胚期的胚胎进行照射,均引起各期胚胎的畸变,这些畸变大多数是初孵仔鱼的头部、尾部、胸腔和背脊的变形。实验结果表明,天使鱼各个发育阶段的胚胎畸变率随着激光辐照剂量的改变而发生变化。     

激光辐照鱼类胚胎导致畸变

红激光(He—Ne,632.8nm)辐照金鱼(Carassius aurafus)受精卵和怀胎孔雀鱼(Poecilia reticulata),导致两种鱼胚胎畸变。金鱼胚胎在某一发自阶段的畸变率随辐照剂量的增加而提高;在同一剂量辐照下,不同发育期的胚胎畸变率各异,胚胎发育初期畸变率最高,此后随胚胎发育过程畸变率下降。经He—Ne激光辐照的孔雀亲鱼也能生产畸形仔鱼。在实验中还比较了红激光(632.8nm)和紫激光(N_2337.1nm)辐照金龟卵致变效果。     

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.     

低强度He-Ne激光照射人外周血淋巴细胞两组试验结果的分析

本文采用染色体畸变 (chromosomalaberration CA)试验和微核 (micronucleus)试验两种方法对低强度He Ne激光辐照育龄妇女外周血淋巴细胞。激光能量密度分别为 14.31J cm2 (辐照 5′)、2 8.6 2J cm2 (辐照 10′) ,5 7.2 4J cm2(辐照 2 0′) ,114.5 2J cm2 (辐照 40′)。照射血样后 ,染色体畸变试验检测其淋巴细胞染色体畸变率 ,激光照射及空白对照组 ,血样染色体畸变率分别为 4.2 9‰、3.96‰、3.81、3.5 9‰和 4.19‰ ,X2 检验无显著意义 (P >0 .0 5 )。阳性对照丝裂霉素MMC处理的血样淋巴细胞CA率平均为 14.41‰ ,明显高于激光照射和空白对照组 ,X2 检验有显著差异(P <0 .0 1)。微核试验检测结果 ,微核染色体分别为 1.0 2‰ ,1.17‰ ,1.18‰ ,1.31‰和 1.19‰对照 ,经统计分析激光照射各组与对照组微核率均在 2‰以下 (P >0 .0 5 ) ,属正常人体微核范围内。结果显示两组试验监测诱变均具有一致性。证明He Ne激光辐照人体细胞对染色体无致畸效应。且表明He Ne激光在治疗范围内应用安全、有效、不会对不体造成危害。     

激光对青蛙卵作用的生物效应

用He—Ne激光分别照射青蛙的卵子、精子和早期胚胎,在相同的激光剂量条件下,观察不同激光照射时间对胚胎发育的影响。通过电镜观察卵膜的超微结构,分析激光作用于细胞的刺激作用机制。     

低强度He-Ne激光辐照人体外周血对淋巴细胞染色体影响的研究

本文通过低强度 Ｈｅ Ｎｅ 激光以能量密度分别为 １４３１ Ｊ／ｃｍ ２ （辐照 ５′）、２８６２ Ｊ／ｃｍ ２ （辐照 １０′）、５７２４ Ｊ／ｃｍ ２ （辐照 ２０′）１１４５２ Ｊ／ｃｍ ２ （辐照 ４０′）照射人体外周血后,检测其淋巴细胞染色体畸变率（ Ｃ Ａ）,激光照射血样（能量密度由低到高）及未照射血样 Ｃ Ａ 分别平均为 ４２９‰、３９６‰、３８１‰、３５９‰、４１９‰, Ｘ２ 检验无显著差异（ Ｐ＞ ００５）,说明低强度的 Ｈｅ Ｎｅ 激光辐照人体细胞对细胞染色体无致畸变效应。而且随着能量密度的增大,染色体的畸变率有降低的趋势,因此认为是低强度的 Ｈｅ Ｎｅ 激光促使细胞内分子的相互作用和能量转换,从而使染色体损伤的修复增强,其机理有待于进一步的研究。     

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.     

应用激光扫描共聚焦显微镜FRAP技术研究兔早期胚胎发育中细胞间隙连接介导通讯

本研究应用激光扫描共聚焦显微镜的光漂白恢复技术(FRAP)分析兔早期胚胎卵裂球之间通过间隙连接介导的细胞通讯(GJIC)。研究结果发现,用强激光分别将4-细胞期胚胎、异裂胚胎和8-细胞期胚胎的一个卵裂球荧光光漂白后,经过15分钟的荧光恢复,4-细胞期胚胎的光漂白恢复率为17.8％,异裂胚胎的光漂白恢复率为23.7％,二者之间没有明显的差异;8-细胞期胚胎的光漂白恢复率为78.2％,与前二者之间存在明显的差异。推测兔早期胚胎卵裂球细胞间隙连接建立的时间在8-细胞阶段,胚胎卵裂球间隙连接通讯可能是兔胚胎正常发育的重要条件。     

Abnormal chromosome segregation at early cleavage is a major cause of the full-term developmental failure of mouse clones

To clarify the causes of the poor success rate of somatic cell nuclear transfer (SCNT), we addressed the impact of abnormalities observed at early cleavage stages of development on further full-term development using 'less-damage' imaging technology. To visualize the cellular and nuclear division processes, SCNT embryos were injected with a mixture of mRNAs encoding enhanced green fluorescent protein coupled with α-tubulin (EGFP-α-tubulin) and monomeric red fluorescent protein 1 coupled with histone H2B (H2B-mRFP1) and monitored until the morula/blastocyst stage three-dimensionally. First, the rate of development of SCNT embryos and its effect on the full-term developmental ability were analyzed. The speed of development was retarded and varied in SCNT embryos. Despite the rate of development, SCNT morulae having more than eight cells at 70h after activation could develop to term. Next, chromosomal segregation was investigated in SCNT embryos during early embryogenesis. To our surprise, more than 90% of SCNT embryos showed abnormal chromosomal segregation (ACS) before they developed to morula stage. Importantly, ACS per se did not affect the rate of development, morphology or cellular differentiation in preimplantation development. However, ACS occurring before the 8-cell stage severely inhibited postimplantation development. Thus, the morphology and/or rate of development are not significant predictive markers for the full-term development of SCNT embryos. Moreover, the low efficiency of animal cloning may be caused primarily by genetic abnormalities such as ACS, in addition to the epigenetic errors described previously.     

Laser microbeam-induced DNA damage inhibits cell division in fertilized eggs and early embryos

DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development.     

Cytological Studies on the Embryogenesis of Hybrids between Oryza sativa L.(Pennisetum sp.

(1) The pollen grains of Pennisetum can germinate normally on the stigma of rice and the pollen tubes can grow into the style and enter the embryo sacs. However, the process of double fertilization is slow and more or less abnormal and phenomenon of simple fertilization often occurs. (2) It has been found that in the majority of cases the development of the embryos is slow and stays long in the stage of globular embryos, thus, the differentiation of the embryos is very difficult and degeneration of the embryos appears many times. Simple differentiation was observed only in some embryos during 16–24 days after pollination. Normal differenting and developing embryos were not observed. The cause of the degeneration of the embryos is related to the state of endosperm development and also to the non-coordination of the genomes of both parents. (3) The development of the endosperm is abnormal. The change from the free nuclei into the cells in the endosperm is delayed as late as the 8th day after pollination. The whole endosperm tissue is composed of the cell masses which are quite different both in shape and function, a part of these endospemn cells lacks the ability to synthesize starch. The disintegration of the endosperm could be frequently observed during their development. (4) A lots of starch are accumulated in the nucellar cells near the antipodals, It is shown that there was some metabolic confusion resulted from the crossing in the embryo sacs. Based on the above mentioued results the authers consider that the failure of producing seeds by crossing is at least related to the nutrient condition which are essential for the development of embryos. If embryo culture technique is employed at the early stage of the embryo development the hybrid seeds could be obtained.     

The Fine Structural Observation on the Development of Embryo and Endosperm in the Intergene- rfc Crosses of Fadish ♀ (Raphanus sativus L.) Cabbageo ♂ (Brassica oleracea L.)

The present report deals with the structural abnormalities and abortion of the endosperm, and the related abnormal development of the embryos in the intergeneric crosses of radish (Raphanus sativus L.) ♀ with cabbage (Brassica oleracea L.) ♂. The weak development of ER, the occurence of starch grains in chloroplasts, the curious distribution of chloroplasts around the nuclei, and the earlier formation of cellular endosperm are some primary structure abnormalities of endosperm and they may indicate the poor development, low metabolic activities, and precocious growth in the hybrid endosperm. The endosperm abortion, which starts from chalazal end endosperm, comprises the damage of both structure and function of ER membrane, and the disintegration of nuclei and organelles, or even death. The hybrid embryos degenerated very early in 2-cell stage or even zygote stage in case the endosperm Aborted early. In case of endosperm late-aborted, the hybrid embryos grew for quite a long period of time but become slow down as the endosperm showed abnormal development and started to abort. As the results, the embryos were smaller, the embryo propers and the suspensors showed no growth in step with one another, and the embryo cells appeared structural abnormalities, and finally degenerated and aborted.     

A quantification model for apoptosis in mouse embryos in the early stage of fetation

Apoptosis is the most important inducement and modulator for embryos in the early stage of fetation, i.e. after the 8-cell stage, mostly the morula and blastula stage, to proceed to the stage of nonlinear development. Using a two-photon laser scanning microscopy (TPLSM) system, we obtained 3-dimensional (3D) fluorescent images of preimplantation mouse embryos. A model for quantification was established. The statistical results for the spatial location of apoptosis bodies in embryos was obtained following image processing, as well as investigation of the kinetics of apoptosis. It was found that most (70%) apoptosis occurred in the trophectoderm, and the departure between the centroid and geometric center of embryos had a step transition when embryos developed into the 32-cell stage, which was consistent with the theoretical prediction that the blastocele would induce a symmetry break of the distribution of cells in embryos.