鱼类染色体转移—仙台病毒融合胚胎细胞核移植的初步研究

细胞杂交技术近二十年来取得了巨大的成 就,现已被广泛地应用于医学和生物学各个领 域[4,51。但是体细胞杂交只能得到杂交的细胞, 还不能得到杂交的个体,至少在动物界是如此。 在植物方面已经能通过原生质体的融合产生杂 交细胞,再通过杂交细胞的培养产生新的再生 植株〔2,71。而动物的繁殖只能由生殖细胞通过 胚胎发育才能产生新的个体。虽然不少人曾做 了许多尝试,想通过卵子和体细胞人工融合的 方法产生杂种,但迄今还没有成功［[8-10,1310童第 周等11,11,121在鱼类细胞核移植方面做了不少工 作,利用鱼类囊胚细胞的核,移植到不同属和不 同亚科的去核未受精成熟卵内,得到了几种核 质杂种鱼。这些工作不但证明了鱼类早期囊胚 细胞具有发育的全能性,同时也说明利用动物 早期胚胎的体细胞核育种的可能性。那么用囊 胚细胞的融合引进外来的染色体以及改造原有 细胞的遗传组分,然后把融合的囊胚细胞的核 移植到去核的未受精的成熟卵里去,是不是能 产生具有新的遗传性状的个体呢？本实验正是 基于这样的目的进行的。首先我们使用同源的 囊胚细胞融合,然后移植核,以了解：(1)鱼类 囊胚细胞融合的条件;(2)融合后的囊胚细胞核 移植是否能促使卵子发育;(3）发育的胚胎染色 体组成如何？以便为导人异源染色体创造条件。     

鱼类培养细胞核发育潜能的研究

本文用细胞核连续移植方法,从鲫鱼囊胚细胞的继代培养细胞,获得一尾存活达三年之久的移核鱼,但性腺未分化,不育。并从性成熟的鲫鱼短期培养肾细胞,获得一尾完成发育的性成熟的成鱼。实验结果提示鱼类囊胚细胞的继代培养细胞核和已分化的成鱼体细胞核仍具发育的全能性,为用细胞核移植方法进行鱼类体细胞育种的可能性提供了依据。     

鼠兔核质杂交胚胎早期发育的研究

细胞核移植技术已被证明是研究发育中核质相互关系的非常重要的手段之一,电融合技术也是近十年发展起来的新型细胞融合技术。本实验运用这两项技术,进行了鼠、兔目间核质杂交实验,小鼠8-细胞核在激活的兔去核卵母细胞中,发生了染色体超前凝聚及核膨胀,融合卵移植到小鼠输卵管4.5天后,冲洗出,有5.4%的重构卵发育到囊胚期,通过染色体检查,囊胚细胞中均为小鼠染色体,其中一个囊胚为正常小鼠核型(2 n=40,XX)。通过本实验,我们认为:鼠兔远缘核质杂交胚胎的早期发育是可能的。     

不同纲动物间的细胞核移植——将小鼠细胞核移到泥鳅细胞质中

我们以前的细胞核移植工作^[1,2]表明：移核卵早期胚胎发育模式(主要指卵裂速度及卵裂模式)与受体卵相同,而与供体核种类无关;供体与受体之间亲缘关系的远近及染色体数目的差异程度,对移核卵囊胚以后的发育有重要影响,但对不同组合间细胞核移植所得囊胚比例影响较小。由此我们可以假定,移核卵最初发育的启动及囊胚以前的发育主要与受体卵有关,而和供体与受体之间亲缘关系的远近关系不大。为了进一步验证这一理论并寻找除染色体数目⑵以外其它影响移核卵囊胚以后发育的因素,在本实验中,我们选择了小鼠(图版Ⅰ—A)和泥鳅(Ⅰ—B)即不同纲间动物作为细胞核移植的材料。     

中华大蟾蜍和花背蟾蜍间移核胚胎早期发育中染色体变化的研究

本研究以中华大蟾蜍和花背蟾蜍间正反核、质组合的移核胚胎为材料,对其三个不同发育阶段(囊胚晚期、原肠早期、原肠晚期)的移核胚胎染色体进行了组型分析。结果表明,无论以中华大蟾蜍囊胚细胞核作供体,花背蟾蜍去核未受精卵作受体的移核胚胎,还是相反组合的移核胚胎,在三个不同发育阶段,其染色体数目和形态均有变化,即:(1)表现出数目上有规律性的减少,一般减少2条、3条和4条;(2)出现异型染色体,如染色体断裂和双缢痕染色体;(3)表现出比较恒定的异常组型,即缺失第3染色体,多第4、5、6另染色体。在三个发育时期的分裂中期象中,具有2n=22者,中核组占40.3％-41.3％,花核组占52.3-53.1％。     

不同纲动物间的细胞核移植——将小鼠细胞核移到泥鳅细胞质中

我们以前的细胞核移植工作［１,２］表明：移核卵早期胚胎发育模式（主要指卵裂速度及卵裂模式）与受体卵相同,而与供体核种类无关;供体与受体之间亲缘关系的远近及染色体数目的差异程度,对移核卵囊胚以后的发育有重要影响,但对不同组合间细胞核移植所得囊胚比例影响...     

兔体细胞核移植的初步研究

实验以兔胎儿成纤维细胞为核供体,对兔体细胞核移植技术的融合,激活和发育等环节进行了初步研究。实验通过比较不同电场强度对兔2细胞胚胎卵裂球融合以及卵母细胞激活的影响,证实200和260V/mm的电场强度可有效地诱导2细胞胚胎的融合和兔卵母细胞的孤雌激活。然后将200和260V／mm电场强度用于体细胞核移植,融合率分别为44．4％和48．4％,卵裂率分别为58．8％和53．8％,桑椹胚／囊胚发育率分别为5．9％和5．5%。但112枚核移植胚胎移植到5只受体后没有幼子出生。结果表明,实验中所建立的程序至少可以支持兔体细胞克隆胚胎的早期发育。     

金鱼胚胎细胞超低温保存及其发育能力的研究

将金鱼囊胚期的细胞分离后,保存于-196℃液氮中,并对保存后化冻的细胞用细胞核移植的方法检测了其细胞核发育的能力。本文首次证明了经冷冻后的细胞核被移至去核卵中能发育成正常的个体。并证明在冷冻液中加入胶原蛋白可以提高化冻后细胞的存活率。本实验的结果为长期保存端黄卵的各种鱼类的基因库提供了新的途径。     

鼠兔核质杂交胚胎早期发育的研究

细胞核移植技术已被证明是研究发育中核质相互关系的非常重要的手段之一,电融合技术也是近十年发展起来的新型细胞融合技术也是近十年发展起来的新型细胞融合技术,本实验运用这两项技术,进行了鼠、兔目间核质杂交实验,小鼠８－细胞核在激活的兔去核卵母细胞中,发五了染色体超前凝聚及核膨胀,融合卵移植到小鼠输卵管４．５天后,冲洗出,有５．４％的重构卵发育到囊胚期,通过染色体检查,囊胚细胞中均为小鼠染色体,其中一个囊     

牛体细胞核移植显微操作环节的优化

本研究从牛卵母细胞去核方法(纺锤体观测仪法&Hoechst33342染色法)、供体细胞核引入去核卵细胞质的方法(卵细胞质注射法和电融合法)和重构胚胎电融合(3组参数)等3个环节对牛体细胞核移植的显微操作过程及相关参数进行了筛选优化。以核移植胚胎的卵裂率、囊胚发育率作为检测指标,对不同的方法所获得的克隆胚胎的卵分裂率与囊胚发育率进行比较,最后筛选获得1个优化的牛体细胞核移植操作程序,即采用Spindle view系统对牛卵母细胞进行去核操作,将供核体细胞注射到卵周隙,然后通过电融合法将供体核引入去核卵细胞质(电融合参数为1.9kV/cm,脉冲时程10μs,方波2次间隔2s)。以此核移植程序进行牛体细胞核移植实验,自获得克隆胚胎中筛选80枚优质囊胚移植到33头受体牛子宫内,最后2头母牛产下2头克隆牛犊,结果表明利用该优化的显微操作环节进行牛体细胞核移植可以获得体细胞克隆牛犊。     

IMAGE ANALYSIS OF CHROMATIN IN CELLS OF PREIMPLANTATION MOUSE EMBRYOS

Mouse two-celled embryos and blastulae were Feulgen stained and the DNA content of their nuclei was measured with an integrating microdensitometer. The cells considered on the basis of their nuclear DNA content to be in G₁, S, and G₂ phases of the cell cycle were selected and their total chromatin area and chromatin areas at different gray levels were measured by the image analyzing computer, Quantimet. The measurements were aimed at quantitation of several features of the chromatin morphology of cells in different functional states. The total area of chromatin was found to increase, and the mean density of chromatin to decrease, from the G₁ to the G₂ phase of the cell cycle in both two-celled embryos and blastulae. The area of chromatin decreased, and the mean density of chromatin increased, as embryos developed from two-celled to blastula stage. It was concluded that nuclear morphology in preimplantation mouse embryos depends on both the phase of the cell cycle and the stage of development. The method of image analysis described was found to be useful for quantitation of changes in chromatin morphology.     

FORMATION OF JOINED LARVAE IN THE STARFISH, ASTERINA PECTINIFERA

A method for joining larvae in the starfish, Asterina pectinifera , is presented. Any number of embryos are stably united by simple contact of the cell clusters (descendents of individual denuded eggs) before reaching the early blastula stage (ca. 6 1/2 hr after insemination at 21°C). Embryos do not combine with each other after closing into hollow blastulae. This is considered to indicate the transformation of the cell cluster from a mere collection of cells to an individual, multicellular system. Biological meanings of the fertilization membrane are discussed.     

Transplantation of somatic nuclei into activated teleost eggs (the example of the loach, Misgurnus fossilis L.)]

Some peculiarities of the technique of nuclear transplantation for teleostean fishes are described, the loach (Misgurnus fossilis) taken as an example. The cells of the late high blastula were used as donors and the activated non-enucleated and enucleated (by X-rays, 20 kR) eggs of the loach as recipients. Following the nuclear transplantation in the activated non-enucleated eggs, 33 (out of 128) normal blastulae were obtained, one of which developed until hatching. Following the nuclear transplantation in the activated enucleated eggs, 34 (out of 251) blastulae were obtained, 6 embryos hatched and 2 larvae attained the stage of active feeding.     

Spectrofluorometric assay for the quantitation of cell-tissue electrofusion.

Fluorometric assay for quantitating electrofusion, FAQE, was developed to measure the number of cells fused to intact tissue. The fluorescent vital dye hydroethidine is used in this method. The fluorescent intensity detected in the cell tissue hybrids is proportional to the number of individual cells fused. The number of cells fused was determined after fusion by lysing the epithelial layer with 0.2% sodium dodecyl sulfate and the supernatant fluids were measured in a spectrofluorometer and compared to established standard curves. The mean number of cells fused, in five separate experiments, was determined to be approximately 5000. All the experimental corneas had approximately the same number of fused cells with less than 10% variation. In addition, the technique was used to demonstrate an increase in the number of cells fused when multiple fusions were applied to the cell-tissue electrofusion system. These results demonstrate that FAQE can be utilized to quantitatively analyze the fusion yields.     

Elevation of Cyclic AMP-Dependent Protein Kinase Activity during Migration of Primary Mesenchyme Cell in Sand Dollar Blastulae

Concetration of intracellular cyclic AMP (cAMP), and activities of adenylate cyclase and cAMP-dependent protein kinase were examined in swimming and mesenchyme blastulae and primary mesenchyme cells (PMCs) of the sand dollar, Clypeaster japonicus , respectively. In mesenchyme blastulae, the concentration of cAMP increased 45% from that in swimming blastulae. PMCs contained a concentration of cAMP 40% higher than that in whole embryos at the mesenchyme blastula stage. The activity of adenylate cyclase in mesenchyme blastulae was 100% higher than that in swimming blastulae. The activites of cAMP-dependent protein kinase in whole embryos at the above two developmental stages, on the other hand, were quite similar to each other. However, in PMCs the activity of the enzyme was conspicuously higher than that in these embryos, and it reached 190% higher than that in these embryos. Inhibition of cAMP-dependent protein kinase activity by a synthetic inhibitor, H8, caused severe inhibition of PMC migration but it did not exert any effect on PMC ingression. These results suggest that the cAMP-dependent protein kinase activity is involved in PMC migration, but not in PMC ingression.     

Exogut Formation by the Treatment of Sea Urchin Embryos with Ascorbate and α-Ketoglutarate

In sea urchin embryos at the stages from hatch out to the pluteus stage, [¹⁴C]proline incorporation into hot trichloroacetic acid TCA-extractable proteins occurred during an exposure to [¹⁴C]proline for 3 hrs at 20°C. The rate of [¹⁴C]proline incorporation into hot TCA-extractable proteins was higher in gastrulae and plutei than in blastulae. Percentage of [¹⁴C]hydroxyproline residue to whole radioactivity of the hot TCA-extractable proteins was quite low at the blastula stage and increased exponentially during futher development. Production of [¹⁴C]hydroxyproline residue at the blastula stage, as well as at the later stages, was stimulated by ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, and inhibited by α, α'-dipyridyl, an inhibitor of this enzyme. It is also probable that the enzyme in the embryos is not fully activated because of low amounts of activating substances. These suggest that blastulae,…, also have a potency of protocollagen hydroxylation. Blastula kept in sea water containing ascorbateand α-ketoglutarate became undeveloped embryo with large exogut. Gastrula developed normally to pluteus even in the presence of these compounds. The embryos, kept in sea water containing these compounds from fertilization to hatch out, also developed normally. Exogut formation in the embryos treated by these compounds, as well as normal archenteron formation, was inhibited by α, α'-dipyridyl.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

VEGETALIZATION OF SEA URCHIN LARVAE INDUCED WITH cAMP PHOSPHODIESTERASE INHIBITORS

Treatment of sea urchin embryos with cAMP phosphodiesterase (PDE)-inhibitors such as caffeine (4×10⁻³ M), theophylline (8×10⁻³M), or nicotinamide (10⁻²M), at the morula stage for only a couple of hours, yields vegetalized larvae. Most of the embryos treated with these reagents before the morula stage develop to blastulae filled with mcsenchyme-like cells. Almost all embryos at the blastula stage develop normally even if they are treated with a PDE-inhibitor for a considerable period. The rate of ³H-valine incorporation into protein in the morulae is reduced by caffeine and theophylline, but does not decrease in the presence of nicotinamide. Actinomycin D cancels the vegetalizing effect of PDE-inhibitors on the morulae.     

Cellular Events of Wrinkled Blastula Formation and the Influence of the Fertilization Envelope on Wrinkling in the Seastar Patiriella exigua

Like many echinoderms, the seastar, Patiriella exigua has a wrinkled blastula rather than the smooth-walled blastula typical of most phyla. The cellular events of wrinkled blastula formation in P. exigua were documented using light, confocal and electron microscopy. Wrinkled blastulae have a highly infolded epithelium. Prior to wrinkling, the blastomeres are cuboidal with lipid droplets and yolk granules distributed throughout their cytoplasm. During wrinkling, the cells become columnar and the lipid and yolk reserves become redistributed to the basal and apical ends of the cells, respectively. Gastrulae have a tall columnar epithelium, with a basal accumulation of lipid. Interdigitation of numerous cell projections, including short lateral processes, basal lamellipodia and apical filopodia, assists in maintaining epithelial integrity during wrinkling. Apical filopodia have not been observed in other echinoderm embryos. Although 1 M urea caused elevation of the fertilization envelope, the embryos did not expand into the newly-created space. This is suggested to be due to the adhesive properties of the hyaline layer. Embryos removed from their envelope were enlarged with shallower and fewer wrinkles compared with controls. It appears that the integrity of the hyaline layer and fertilization envelope both influence the compact wrinkled profile of P. exigua blastulae.     

Species-specific dissociation into single cells of live sea urchin embryos by fab against membrane components of Paracentrotus lividus and Arbacia lixula

The species and stage specificities of membrane components active in promoting reaggregation of cells dissociated from embryos of the two Mediterranean sea urchin species Paracentrotus lividus and Arbacia lixula have been examined. Membrane proteins extracted with butanol either from purified membranes or from dissociated cells without significant reduction of viability promoted reaggregation of both the homologous and heterologous species. Extracts from plutei and blastulae were equally effective in promoting reaggregation of blastula cells. By contrast, Fab's prepared from IgG raised against these extracts or purified membranes are strictly species specific because they prevent reaggregation of cells and actively dissociate live embryos of only the homologous species. No corresponding stage specificity of the Fab was observed: Fab against extracts from blastula embryos also caused dissociation of plutei. Antigenic analysis of the extracts by the Ouchterlony test revealed the presence of components specific for each species as well as others common to both.