鱼类卵子发育能力的进一步研究

作者中的二人[Tung,and Tung.(童第周与叶毓芬)1944;Tung,Chang,andTung.(童第周、张致一与叶毓芬)1945]曾以分割的方法,研究金鱼(Carassiusauratus)卵子的发育能力。他们将2至8细胞时期的金鱼卵子,在去膜后,用玻璃针,按第一次或第二次的分裂平面,将整个卵子分割为二半。每半含有半数分裂球,一半卵黄,和一半未流入分裂球的细胞质。分割后,每半都能继续发育,但产生下列6种不同的情形:(1)二半都发育为完整的,一半大小的胚胎;(2)一个为完整的胚胎,另一个为不完整的胚胎;(3)一个为完整的胚胎,另一人为囊胚;     

分割兔胚获得半胚发育的小兔

哺乳动物胚胎分割的研究,近十年来有很大进展。继小鼠(1970年)首次分割成功后,大鼠、猪、牛、山羊和绵羊等胚胎分割亦已成功,得到由半胚发育的后代。至今已有人工制造的同卵双胎、三胎及四胎,分割方法亦由复杂到简单。特别是最近一些研究结果证实,多种动物的晚桑椹或囊胚期胚胎分割后,半胚不需要再     

胚胎分割的研究进展

近二十年来 ,通过分割哺乳动物的早期胚胎来人工产生遗传上完全相同的同卵双胎或多胎动物 ,在绵羊、牛、小鼠、大鼠、马、兔、猪等哺乳动物中已广为开展〔１～１４〕。为生产上提供了具有优良品种（产肉、产奶量高等）的动物 ,同时 ,还给发育遗传学、生理学等领域提供了进行科学研究的宝贵材料。所谓胚胎分割系指用人工方法将一个植入前胚胎分割成两个或多个部分 ,每一部分移植后可发育成一个正常个体。早在１９５９年,Ｔａｒｋｏｗｓｋｉ〔１５〕发现一半卵裂球已被破坏的２ -细胞小鼠胚胎可以发育成囊胚 ,并出生了小鼠。其研究结果表明 :…     

核移植技术研究进展

核移植(nuelear transplation,NT)是将动物早期胚胎或体细胞的细胞核移植到去核的受精卵或成熟卵母细胞中、重新构建新的胚胎,使重构胚发育为与供核细胞基因型相同后代的技术过程,又称动物克隆技术。广义的胚胎克隆技术还包括胚胎分割和卵裂球培养,通常所指的胚胎克隆技术是指狭义概念,即核移植技术。1938年Spmann在所有胚胎细胞都具有与受精卵完全相同、拥有潜在发育全能性的细胞核基础上提出了细胞核移植的概念[‘1。早期核移植实验是在变形虫、蛙、爪蟾、非洲爪蛙等两栖类和鱼类上进行的核质关系研究〔“一“〕,随着胚胎技术的不断进步…     

奶牛ZFY、ZFX基因片段的克隆及测序

奶牛的早期胚胎性别鉴定是奶牛胚胎分割及移植技术产生较大经济效益的前提,用PCR技术进行早期胚胎性别鉴定具有准确、快速、灵敏度高的特点.利用人和鼠的性别分化相关的DNA序列ZFY、ZFX基因序列设计的引物对公牛和母牛的染色体DNA、PCR扩增,将扩增产物定向克隆到pUC118上,获得ZFY、ZFX转化子,并测定了ZFY、ZFX基因的序列,发现两者同源性达88.2%,以此为基础可设计引物和探针,以PCR方法进行高灵敏度的奶牛性别鉴定.     

封面及有关照片说明——切割牛胚胎得到孪生牛

美国科罗拉多州立大学的科学家发明了一种切割和移植胚胎的技术,从一个胚胎产生二头动物。用这种技术可以增加良种动物的繁殖率,并可得到用于实验室研究的遗传学上一致的孪生动物。上图1为武田哲夫博士在福特·克林斯学校胚胎移植实验室里演示这种技术。他正准备把一个中空的玻璃吸管(他手指所指示的)插到一个放置一周牛胚胎的培养皿中。上图2是通过显微镜听见到的情况。他固定着一个将被分割的胚胎。用左侧的吸管借助吸力固定着胚     

斑马鱼胚胎彩色图像生理结构分割

斑马鱼是一种当今应用十分广泛的模式生物,研究其实验图像的信息自动提取具有较高的应用价值。药物培养斑马鱼胚胎在发育成长过程中的生理影响的定量分析,很难通过人工肉眼观察的方式得出较为准确的指标值,通常要借助于计算机。斑马鱼图像定量分析的前提基础就是可以对斑马鱼胚胎图像,按照斑马鱼的生理结构即头部、躯干和卵黄三个部分在图像中分割开。但是实际情况是斑马鱼药物实验中特定药物的实验组数量有限,不能通过学习训练等机器学习的方式分割,只能使用图像处理建模分割。本文使用距离变换结合分水岭算法、减法聚类结合K-means聚类算法以及减法聚类结合漫水填充算法分别对图像进行语义分割,发现减法聚类结合漫水填充算法的分割效果达到了满足研究所需的生理结构分割的目的,为后续医学研究中的定量分析奠定了较好的基础。     

生物学家对于建立人-鼠嵌合胚胎的不同看法

在纽约举办的讨论关于人类胚胎研究标准的论坛上 ,一些生物学家认为人类胚胎干细胞应该注入鼠的胚胎中以检测它们的临床应用前景。ＡｌｉＢｒｉｖａｎｌｏｕ (ＲｏｃｋｅｆｅｌｌＵｎｉｖｅｒｓｉｔｙｉｎＮｅｗＹｏｒｋ)等认为 ,在检验人类胚胎干细胞的多能性时需要这种嵌合式的胚胎。而胚胎干细胞将来的临床应用正是利用了这种多能性 ,即这一种类型的细胞可以向不同方向发育而发挥不同的功能。伦理学反对将人类细胞注入人类胚胎的实验。所以研究者认为应该将人类的胚胎干细胞注入鼠的胚胎中 ,继而植入雌性鼠体内让其生长发育 ,然后在发育…     

电穿孔介导小干扰RNA高效转染小鼠附植前胚胎

使用Cy3标记的阴性对照小干扰RNA(siRNA)转染小鼠附植前胚胎,建立向小鼠附植前胚胎导入siRNA的电穿孔方法。通过控制透明带弱化程度、电压、脉冲时间和脉冲次数等条件,采用不同参数组合并结合使用不同介质作为电转缓冲液将Cy3标记的阴性对照siRNA转染小鼠附植前胚胎。在荧光倒置显微镜下,观察胚胎的存活率、siRNA转染率以及阳性转染存活胚胎的囊胚发育率。结果显示小鼠附植前胚胎在使用台氏液消化胚胎透明带10 s后,以opti-MEM作为电转缓冲液,电穿孔参数设置为30 V,1 ms,3次的条件下取得最佳转染效果。总之,电穿孔方法可实现siRNA简便、高效地转染小鼠附植前胚胎。     

小鼠胚胎徒手分割技术的研究

目的　研究不同分割液和分割前胚胎去致密化与否对昆明白系小鼠的桑椹胚和囊胚的徒手分割以及分割后胚胎移植的影响。方法　在mPBS、1 2 5 %蔗糖液和进口分割液三种不同分割液中对桑椹胚和囊胚进行徒手分割。结果和结论　在蔗糖液与进口分割液中分割桑椹胚 ,成功率显著高于mPBS处理组 (6 9 5 3%、77 4 0 %vs5 6 82 % ) (P <0 0 5 ) ,而半胚的囊胚发育率及囊胚细胞数三组差异均无显著性 (P >0 0 5 ) ;在蔗糖液与进口分割液中分割囊胚 ,分割后半胚培养的囊胚发育率显著高于mPBS处理组 (72 38%、74 2 9%vs 5 6 2 0 % ) (P <0 0 1 ) ,而分割成功率及囊胚细胞数三组差异均无显著性 (P >0 0 5 ) ;各处理组半胚的囊胚发育率及囊胚细胞数都显著低于对照组体外培养桑椹胚的囊胚发育率 (98 70 % )和囊胚细胞数 (6 3 6 7± 5 78) (P <0 0 1 ) ;桑椹胚经去致密化处理后分割 ,其分割成功率显著高于未处理组 (82 90 %vs 5 6 6 0 % ) (P <0 0 1 ) ,处理组半胚培养的囊胚发育率也显著高于未处理组 (73 80 %vs 4 6 80 % ) (P <0 0 1 ) ;共移植 1 4 2枚 2分胚形成的囊胚移植于 1 1只受体 ,其中 3只妊娠 ,分别产仔 2只、3只和 4只     

Quick-splitting of bovine embryos

Described is a simplified method of bovine embryo bisection amenable to on-farm embryo transfer. Using a microblade operated by a hand-held micromanipulator, Day 7 bovine embryos were bisected while in the zona pellucida. With a vertical motion, the embryo was pinned between the blade and the bottom of a plastic petri dish and bisected. Demi-embryos were transferred nonsurgically (without zonae pellucidae) into synchronized recipients. Pregnancy rates were normal with 5 13 (38%) and 9 20 (45%) of recipients confirmed pregnant 70 to 80 d after receiving either twin or single half embryos, respectively. This compared to 12 28 (43%) of recipients becoming pregnant from transfer of whole embryos. These data confirm that bovine demi-embryos do not need zonae pellucidae on Day 7 and that simplified field methods of bisection give normal pregnancy results.     

Somatic cell cloning without micromanipulators

Until now, micromanipulators have been regarded as indispensable for somatic cell nuclear transfer. This paper describes an improved zona-free nuclear transfer procedure with manual bisection of oocytes, selection of cytoplasts by Hoechst staining, and two-step fusion of somatic cells from primary granulosa cell cultures with two cytoplasts. Blastocyst rates in the three systems tested for zona-free embryo culture were 0%, 18%, and 36% for microdrops, well of the wells (WOW system), and microcapillaries (GO system), respectively. This simple, rapid, and inexpensive procedure may become a useful alternative to the existing techniques for somatic cell nuclear transfer for large-scale application of the technology.     

Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma

Embryos of the indirect developing sea urchin, Heliocidaris tuberculata, and of Heliocidaris erythrogramma which develops directly without the formation of a pluteus larva, were bisected at the two- and four-cell stages. Paired half-embryos resulting from the bisection of H. tuberculata embryos along either the first or the second cleavage plane develop identically into miniature prism stage larvae. As in other indirect developing sea urchins, no differential segregation of developmental potential takes place as a result of the first and second cleavage divisions. Although half-embryos resulting from bisection along the second cleavage plane differentiate all cell types and develop equivalently in H. erythrogramma, the isolated first cleavage blastomeres do not. One of these two cells always forms significantly more mesodermal and endodermal cells. These patterns of differentiation are consistent with fate-mapping studies indicating that most mesodermal and endodermal cells are derived from the prospective ventral blastomere. Therefore, a differential segregation of developmental potential takes place at the first cleavage division in H. erythrogramma. When embryos of H. erythrogramma were bisected during the eight-cell stage, isolated tiers of animal blastomeres typically formed only ectodermal structures including the vestibule, whereas vegetal embryo halves formed all differentiated cell types. We propose that animal-vegetal cell determination and differentiation takes place along an axis which has been shifted relative to the pattern of cell cleavages in the embryos of H. erythrogramma. Vegetal morphogenetic potential for the formation of mesodermal and endodermal structures has become more closely associated with the prospective ventral side of the embryo during the evolution of direct development in Heliocidaris.     

Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro

The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively.     

Splitting and biopsy for bovine embryo sexing under field conditions.

Improvements on embryo micromanipulation techniques led to the use of embryo bisection technology in commercial embryo transfer programs, and made possible the direct genetic analysis of preimplantation bovine embryos by biopsy. For example, aspiration and microsection, allow bovine embryos sexing by detection of male-specific Y-chromosome in a sample of embryonic cells. We report on the application of the methodologies of splitting and biopsy of bovine embryos in field conditions, and on the results of embryo sex determination by the polymerase chain reaction (PCR). Pregnancy rates achieved with fresh bisected or biopsied embryos (50 to 60%) were similar to the fresh intact embryos (55 to 61%). The PCR protocol used for embryo sexing showed 92% to 94% of efficiency and 90 to 100% of accuracy. These results demonstrate these procedures are suitable for use in field conditions.     

Monozygotic twinning in rhesus monkeys by manipulation of in vitro-derived embryos

The nonhuman primate is a relevant model for human disease that can be used for diverse biomedical investigations. The ability to propagate a founder animal by application of assisted reproductive technologies is pressing, but an even greater need in many studies is access to genetically identical animals. In an effort to create genetically identical monkeys, we evaluated two approaches to monozygotic twinning; blastomere separation, and blastocyst bisection. Embryos were produced by intracytoplasmic sperm injection of oocytes recovered following controlled ovarian stimulation. The quality of demiembryos produced in these efforts was evaluated by quantitating the efficiency of creating identical pairs for embryo transfer, by morphological assessment, by the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) in the blastocyst, and by the outcome of embryo transfer to synchronized host animals. Pairs were produced in high yield (85%-95%) by both twinning methods. Demiembryos resulting from blastomere separations at the 2- or 4-cell stage grew to blastocysts at the control frequency. Demiblastocysts contained, on average, half the number of cells of the intact controls while maintaining the same ICM:TE or ICM:total cell ratio. The equivalency of demiblastocysts within a set was also evaluated by differential cell counting. Embryo transfers of identical sets led to a 33% clinical pregnancy rate, with two twin pregnancies initiated. Neither pregnancy resulted in term birth of monozygotic twins, but our results are sufficiently encouraging to justify a large-scale twinning trial in the rhesus macaque.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

Growth and reproduction of mice developed from bisected embryos

Embryos from three groups of mice, ICR (I), a synthetic (S), and an F(1) hybrid from C57BL males and S females (F), were used to examine effects of embryo bisection on subsequent viability, postnatal growth and reproduction. In two experiments, Group S females used as recipients received the following combinations of whole (W) and/or bisected (B) embryos: W(I) and W(F), W(I) and B(F), B(I) and W(F), and B(I) and B(F) in Experiment 1 and W(I) and W(S), W(I) and B(S), B(I) and W(S), and B(I) and B(S) in Experiment 2. Eight to 12 embryos of both types were transferred surgically to two horns of the uterus. Overall survival rate of whole embryos (32.6%) was significantly greater (P < 0.001) than that for demi-embryos (13.2%). The gestation period after transfer was significantly longer (P < 0.05) for demi-embryos (17.5d) than for whole embryos (16.4 d). Mice developed from demi and whole embryos were not different in mean body weight at birth and at 21, 42 and 63 d of age. Fertility and litter size at first parity of mice that developed from bisected embryos were comparable with those that developed from whole embryos. Hence, bisection decreased embryo viability, increased gestation period of recipients but did not affect postnatal growth and reproduction.     

Effect of In Ovulo Period on the Differentiation and Regulation of Immature Embryos ofZamia Cultured In Vitro

Monnier, M. and Norstog, K. 1986. Effect of in ovulo periodon the differentiation and regulation of immature embryos ofZamia cultured in vitro—J. exp. Bot. 37: 1633–1642. Depending upon excision chronology, undifferentiated embryosof the cycad Zamia exhibited two types of development when culturedin vitro. (1) Early embryos, excised in July shortly after fertilization,showed considerable growth of suspensors but, at first, theembryos at the distal ends of the suspensors remained unchanged.As culture progressed, these embryos grew isodiametncally withoutorganogenesis. (2) Late embryos, excised in September, althoughalso undifferentiated at the time of explantation, directlyentered a period of organogenesis with rapid initiation of twocotyledons. These embryos, therefore, seemed to have receivedan inductive stimulus during their more prolonged stay in theovule which permitted subsequent differentiation. This organogeneticimpulse was also expressed even when the embryo was longitudinallybisected. Each half-embryo regenerated a complete embryo havingtwo cotyledons. In this instance, the independent developmentof each half-embryo was a phenomenon of regulation. However,when the longitudinal bisection was done on an old and well-differentiatedembryo, which already possessed two cotyledons, each half-embryocontinued to develop as if it remained attached to the missinghalf. Thus well-advanced embryos did not exhibit regulationto the same degree as do younger embryos and bisection of theembryo resulted in the formation of two half-embryos. Key words: Regulation, differentiation, embryo culture, Zamia     

PRC bisection tests

This communication presents a new method for evaluating phase response curves (PRCs). A PRC describes the phase shifts produced in an oscillator by stimuli applied at different initial phase-states of that oscillator. In the PRC bisection tests, we repeatedly cut in half the circular distribution of the initial phase-states of the oscillator when stimuli are given. Empirically, we locate that optimal diameter which best bisects the circular distribution of phase responses into arcs of relative phase advance and phase delay. We compute a D score reflecting the success of the best bisection. The null hypothesis of a random distribution of phase responses by initial phase is tested with a Monte Carlo procedure, which computes D_r scores from random combinations of phase shifts with initial phases, thus determining the probability, given the null hypothesis, that the observed D score was from a random distribution. The bisection procedure can be extended to examine whether stronger phase shifts are produced in one phase response curve than in contrasting curves. Also, the bisection procedure yields an estimate of the inflection point of the phase response curve. A method is given to estimate the power of the PRC bisection test.