Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%). (C) 2002 Wiley-Liss, Inc.

The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach, Misguruns mizolepis Gunthe

Microcystin-LR, a specific and potent hepatotoxin, was tested for its effects oil loach embryo-larval and juvenile development, The results of this study showed that loach embryos were more sensitive when exposed to microcystin-LR at a later than at an earlier stage of development, Juveniles were far less sensitive to MC-LR than were embryos and larvae. Mortality and developmental abnormality were proven to be dose-dependent and to be stage-specific sensitive. Among the abnormal changes noted were: pericardial edema and tubular heart, bradycardia, homeostasis, poor yolk resumption. small head, curved body and tail, and abnormal hatching, Liver and heart were the main targets of microcystin-LR toxicity. Ultrastructural analysis documented a complex set of sublethal effects of microcystin-LR on loach hepatocytes, chiefly including morphological alteration in nuclear and RER of loach liver cells. fit addition, microcystin-LR was lethal to loach juvenile in the subacute (7 days) exposure (LC50) = 593.3 mug/l). (C) 2002 Elsevier Science Ltd. All rights reserved.

beta-catenin in early development of the lancelet embryo indicates specific determination of embryonic polarity

The lancelet (amphioxus) embryo develops from a miolecithal egg and starts gastrulation when it is approximately 400 cells in size, in a fashion similar to that of some non-chordate deuterostomes. Throughout this type of gastrulation, the embryo develops characteristics such as the notochord and hollow nerve cord that commonly appear in chordates. beta-Catenin is an important factor in initiating body patterning. The behavior and developmental pattern of this protein in early lancelet development was examined in this study. Cytoplasmic beta-catenin was localized to the animal pole after fertilization and then was incorporated asymmetrically into the blastomeres during the first cleavage. Asymmetric distribution was observed at least until the 32-cell stage. The first nuclear localization was at the 64-cell stage, and involved all of the cells. At the initial gastrula stage, however, concentrated beta-catenin was found on the dorsal side. LiCl treatment affected the asymmetric pattern of beta-catenin during the first cleavage. LiCl also changed distribution of nuclear beta-catenin at the initial gastrula stage: distribution extended to cells on the animal side. Apparently associated with this change, expression domains of goosecoid, lhx3 and otx also changed to a radially symmetric pattern centered at the animal pole. However, LiCl-treated embryos were able to establish embryonic polarity. The present study suggests that in the lancelet embryo, polarity determination is independent of dorsal morphogenesis.

Early expressed genes showing a dichotomous developing pattern in the lancelet embryo

Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate-like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior-posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp-related gene, Bbbmp2/4, and two wnt-related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf-3 beta class genes The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region. respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid-dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling, The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior-specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.

The encysted dormant embryo proteome of Artemia sinica

The possibility of the brine shrimp Artemia to produce dormant embryo (cysts) in diapause is a key feature in its life history. In the present study, we obtained a proteomic reference map for the diapause embryo of Artemia sinica using two-dimensional gel electrophoresis with a pH range of 4-7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected, and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these, 39 spots representing 33 unique proteins were identified, which are categorized into functional groups, including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes. This reference map will contribute toward understanding the state of the diapause embryo and lay the basis and serve as a useful tool for further profound studies in the proteomics of Artemia at different developmental stages and physiological conditions.

中国郁金香属(广义)的系统学研究

郁金香属（Tulipa L.）是世界著名的观赏植物，广泛分布于欧洲、亚洲的温带地区以及非洲的西北部，中亚地区是其分布和多样化中心。该属包括老鸦瓣组、长柱组、郁金香组、毛蕊组、扭药组、鸡冠组和无毛组共七个组，40至150种。老鸦瓣组是东亚特有类群。中国共有郁金香属植物16种，主要分布于西北（新疆）以及中东部地区，其中老鸦瓣组有4个种，占该组全部种类的4/5。长期以来，由于对老鸦瓣组的生物学特性及其地理分布缺乏了解，该类群的归属问题一直都是该属系统学研究中争论的焦点之一。因此，本文以分布于我国的郁金香属作为主要研究对象，通过对其形态学、胚囊发育的比较胚胎学以及分子系统学研究，对老鸦瓣组的系统位置以及上述特征在该属分类中的意义进行了探讨。主要结果如下 1)通过对该属18种植物（包括土耳其的3个种）28个形态性状数据的分支分析，表明广义郁金香属并不是一个单系类群。Tulipa sect. Amana与该属其他四个组（sect. Orithyia、sect. Eriostemones、sect. Leiostemones和sect. Tulipanum）在分支树上各成一支，它们与Lloydia属构成一个大支的三个分支。同时，sect. Amana具有与子房近等长的花柱以及2-3（-4）个苞片等与郁金香属不同的形态特征。因此，我们认为sect. Amana应从广义郁金香属中独立出来，恢复其老鸦瓣属Amana Honda作为属的分类地位。 2)发现了一个新种：Amana kuocangshanica D.Y. Tan et D. Y. Hong。该种与A. erythronioides 和A. anhuiensis近缘，区别在于鳞茎皮内侧光滑无毛，下部叶披针形，自基部向上2/3处最宽，果喙长0.64±0.08 cm。 3）对16种植物叶表皮形态观察的结果表明，老鸦瓣属4个种的叶上表皮细胞为矩形或矩圆形、下表皮为菱形或矩形，垂周壁为直线形或波形，上表皮无气孔或气孔密度较小，这些特征与狭义郁金香属的种差异显著。在狭义郁金香属中，叶表皮特征在种间差异明显，可以作为分种及种间亲缘关系探讨的依据，但在组间没有明显的差异。 4）对19种植物的花粉形态观察表明，Amana属的4种为近椭球形、舟形和肾形， 外壁纹饰网状，网脊浅皱波状，与狭义郁金香属的15种具明显不同。在狭义郁金香属中，花粉外壁纹饰虽然在种间存在一定程度的差异，但对组的划分意义不大。 5）从种皮形态及微形态特征看，在所观察的16种植物中，Amana属的种子小，呈半月形，较厚，种柄明显，胚不易见;种皮纹饰为皱波状或不规则，与狭义郁金香属存在显著的差异。狭义郁金香属的12种在种皮纹饰、网眼大小及形状、网脊宽窄等方面均存在明显的差异，但组间无明显差别，说明这些特征在种的划分上具有一定的分类学意义。 6）对16种植物的胚囊发育过程观察表明：共有6种胚囊发育类型，即Fritillaria type、Drusa type、Tulipa iliensis type、Tulipa tetraphylla type、 Adoxa type和Eriostemones type。其中Tulipa iliensis type为所发现的一种新的胚囊发育类型。Tulipa iliensis、T. heterophylla和T. heteropetala3个种具有两种胚囊类型。在郁金香属中，胚囊的发育类型具有一定的系统学意义。 7）通过对21种郁金香以及猪牙花属2种植物基于nrDNA ITS区和cpDNA trnL-F 区的序列分析，发现广义郁金香属并非一单系类群，老鸦瓣属为猪牙花属的姐妹群。在狭义郁金香属中，sect. Orithyia、sect. Tulipanum以及sect. Eriostemones得到了该分子系统学分析的支持，而sect. Leiostemones是否成立及其系统关系问题尚有待于进一步研究。

水稻胚囊发育畸变的细胞学和遗传学研究

1．水稻多卵卵器的起源：被子植物的卵器中通常只有一个卵细胞。我们在水稻多胚品系胚囊中观察到二卵卵器和三卵卵器，本研究对其大孢子发生和胚囊发育进行了细胞胚胎学观察，揭示了水稻多卵卵器的起源．观察结果表明，该品系能进行正常的大孢子发生。大孢子母细胞进行正常的减数分裂形成四个大孢子靠近合点端的大孢子发育，其它三个退化。功能大孢子第一次有丝分裂后两个子核被一中央大液泡分隔在胚囊珠孔端和合点端，与此同时胚囊出现不均衡生长，珠孔端迅速膨大，合点端几乎不增大，致使二核末期的胚囊呈倒梨形．紧接着发生第二次有丝分裂，合点端核分裂时纺锤丝与胚囊纵轴平行，而珠孔端核分裂时纺锤丝与胚囊纵轴成4 5度夹角．由此产生的四核胚囊中，合点端一核向胚囊中部或中上部（胚囊珠孔端）迁移，四核胚囊再经一次有丝分裂形成两种类型的核分布偏离蓼型的八核胚囊。一种类型是珠孔端四个核，中部与合点各二个核，在胚囊细胞化过程中，珠孔端四核 分化成四细胞卵器，其中卵细胞和助细胞各二个，中部的二核分化成二极核中央细胞，合点 端的二核形成反足细胞。另一种类型是珠孔端六个核，合点端二个核，在胚囊细胞化过程中， 两端各一核向中部迁移分化成二极核中央细胞，珠孔端剩余的五核分化成五细胞卵器，其 中卵细胞三个，助细胞二个，合点端的一核迅速分裂形成反足细胞． 2．水稻同源三倍体TAR的生殖特性：TAR的单穗结实率平均可达10%，核型分析表明此三倍体产生的后代个体仍为具有36条染色体的三倍体．细胞胚胎学初步观察显示TAR为一具兼性无融合生殖特性的水稻新种质，其胚珠几乎都能进行胚囊的分化，但其中仅有33%的胚囊有较正常的结构，9%的胚囊在散粉前进行胚胎发生，58%的胚囊发育显著异常，表现为极性紊乱、多极核或缺失雌性生殖单位等。 3．水稻亚种间杂种败育的细胞学基础：对普通栽培稻不同品种类型间杂种颖花败育的细胞学基础及雌性败育的过程进行的细胞学研究表明：1）引起杂种颖花败育的原因有胚囊败育，花粉败育、开花时花药不开裂和雌雄异熟．其中胚囊败育而丧失受精能力是引起低结实率的最重要的因素，开花时花药不开裂和雌雄异熟在一定程度上形成了雌雄性细胞时间和空间的隔离屏障。2）杂种植株的所有大孢子母细胞都能进行正常的减数分裂形成四个大孢子，败育主要发生在靠近合点端的功能大孢子分化形成胚囊的早期，有的胚囊母细胞在进行第一次有丝分裂前便萎缩解体，多数能完成一次或二次有丝分裂形成二核或四核败育胚囊．败育的共同特征是无液泡的分化，细胞质少或退化，在败育胚囊残迹部位，解体的珠心细胞和萎缩的胚囊残溃混杂垛叠．已受精的杂种子房没有观察到胚及胚乳发育的异常．籼粳杂种胚囊败育频率较高． 4．籼粳杂种生殖障碍的基因定位：应用具有1 37个标记位点的籼粳杂交窄叶青8号/京系17)F1花药培养获得的127个双单倍体OH）群体构建的R FLP图谱，对控制籼粳杂种颖花败育的基因座位进行了定位研究。结果在第1、3、4、5、6、7、8、1 2染色体上检测到1 0个基因座位，其中第3、12染色体上的2个不育基因位点str3和str12与同一杂交组合F2分离群体中发现的异常分离热点处于相同的染色体区段．stj-6的基因加性效应为负值，有增加籼粳亲和性的作用;其余的不育基因座位皆有增加籼梗杂种不育性的作用． 5．籼粳杂种胚囊败育的遗传分析和基因定位：利用DH系构建的分子图谱及DH系衍生的2个回交群体定位了引起籼梗杂种胚囊败育的2个互补的主效基因esa-l（E1或e1位点）和esa-2（E2或e2位点），它们分别位于第6和第1 2染色体．在不育基因位点，籼稻基因型为EIEle2e2，粳稻基因型为elelE 2E 2，杂交后代中基因型为EIE2，Ele2、elE 2的雌配子体正常发育，携带ele2基因型的雌配子体表现败育．胚囊育性受配子体基因型控制，孢予体遗传背景影响胚囊败育基因的表达.

用共聚焦扫描显微镜检术和酶法分离技术观测观察水稻胚囊发育

本文中以水稻为实验材料，用共聚焦扫描显微镜检术(CLSM)和酶法分离技术观察其胚囊取得好的效果，主要实验结果如下： 1.水稻子房分别用FAA和4%戊二醛固定液固定，经乙醇脱水，然后用冬青油整体透明及丁香油封片。在共聚焦扫描显微镜下，发育中的水稻胚囊显示自发荧光，可分辨出胚囊内部结构。FAA和4%戊二醛两种固定液效果不同，后者效果更佳。用上述实验程序进一步观察拟南芥菜胚囊及水稻和拟南芥菜花粉表明，CLSM可用于快速观察分析水稻和拟南芥菜胚囊发育过程以及水稻胚囊败育情况，但不适用于观察分析其花粉发育过程。 2.水稻子房用FAA、FPA或卡诺氏固定液固定，经乙醇转至蒸镏水，然后置于由纤维素酶和果胶酶组成的酶液中，在37℃保温条件下进行连续振荡酶解，在振荡过程中辅以手工显微操作，由此即可分离出水稻固定胚珠的胚囊。用相差显微镜观察分离的胚囊表明，胚囊整体性及立体感强，观察效果好。在成功分离水稻固定材料胚囊的基础上，初步尝试了水稻新鲜材料胚囊的分离。

拟南芥功能获得突变体sef的研究

植物顶端分生组织中干细胞数量的维持对于侧生器官的发生至关重要。在干细胞的基因调控网络中WUSCHEL (WUS) 是一个关键成员，围绕该基因形成两个反馈调节环，控制分生组织中干细胞群的平衡。 　　论文分析了用激活标签法 (activation tagging) 获得的突变体sef (stem-ecotopic-flowers)，其最大的表型特点是花序轴上产生异位花和幼苗下胚轴增长。本论文就此两个表型产生的机理进行了探索，以期了解WUS基因的新功能。 　　对sef的表型观察发现异位分生组织不仅在花序轴上出现，而且也出现在叶柄、叶片、托叶叶腋内、花梗、花梗腋内以及花器官上。组织切片结果表明花序轴上的异位分生组织起源于已经分化的皮层细胞。对突变体的分子鉴定证明T-DNA是以单拷贝插入到WUS起始密码子上游810 bp处。对插入位点上下游各10 kb的4个基因在花序轴中的表达水平进行了分析，结果表明只有WUS基因的表达量升高，说明增强子只对WUS基因发挥了激活作用，暗示了WUS基因过表达与异位花之间存在某种联系。转35S::WUS的拟南芥幼苗下胚轴与根部出现异位的生长点;WUS被诱导表达的突变体pga6-1花序轴上出现异位花芽，证实sef的表型是由WUS超表达所导致。利用组织原位杂交和RT-PCR分析了WUS、CLAVATA3 (CLV3)、LEAFY (LFY) 与AGAMOUS (AG) 在异位分生组织中的表达模式与表达水平，结果表明WUS、CLV3、LFY、AG在花序轴表皮以下皮层中异位表达。这些结果表明WUS能激活CLV3异位表达，从而在已经分化的皮层中重新产生具有分生组织特征的细胞，同时WUS异位激活AG的表达并使LFY也在这些异位的分生组织中表达，这些分生组织发育方向被LFY与AG所决定，最终发育为异位花器官。 　　sef突变体另外一个突出的表型是幼苗的下胚轴增长。对幼苗期下胚轴以及胚胎4个时期的胚干细胞数进行统计，结果表明下胚轴与胚干细胞数目都呈现出sef比野生型多而wus-1比野生型少的趋势，因此sef幼苗下胚轴增长是由于细胞数目改变引起的。进一步分析发现这种区别是由于胚胎早期（授粉后1~3天）胚干细胞分裂速率的差异所造成的。利用基因芯片杂交分析突变体的基因表达谱，结果发现许多与细胞分裂相关的基因在sef中表达水平升高。RT-PCR证实这些基因在胚胎时期的表达水平升高，说明胚胎早期胚干细胞分裂速率的不同导致了幼苗下胚轴的异常。 　　综上所述，我们的研究结果揭示了sef异常表型的产生的可能机制。在已经分化的皮层中激活标签介导的WUS超表达激活干细胞标志基因之一CLV3和花器官基因AG，并使LFY异位表达，重新产生具有分生组织特征的细胞，这些分生组织的发育方向被LFY和AG所决定，最终发育为异位花。在sef的早期胚胎中，WUS表达增强使细胞分裂相关基因表达水平升高、细胞分裂增快，说明WUS与细胞周期相关基因的调控存在某些联系。 　　本论文的创新之处在于首次提出WUS表达增强能在分化的组织中产生具有分生组织特征的细胞以及WUS调控细胞分裂的结论。 　　

莲（Nelumbo nucifera Gaertn.）胚芽的光合特性及其暗萌发过程中的光合系统建成

被子植物成熟的种子一般不合有叶绿素，但是莲（Nelumbo nucifera Gaertn.）的胚芽却具有鲜明的绿色，本文较详细地研究了莲胚芽不同于一般被子植物叶组织的色素和光台系统组成，并通过对莲胚芽成熟发育过程中的叶绿素合成和光合系统发育进行分析，探讨了莲胚芽光合特性形成的原因，最后对莲胚芽在黑暗中萌发能发育并建成光合系统的现象进行了研究，主要的结果如下： 1，莲胚芽不仅含有叶绿素和光合系统，而且其色素和光台系统组成均与莲叶以及其它被子植物的叶组织不同。莲胚芽的Chla/b值约为0.8左右，远远低于正常高等植物的Chla/b值(～3)：莲胚芽的色素组成中不含有β-胡萝卜素;莲胚芽的光合系统没有电子传递活性，快速荧光动力学测定结果表明莲胚芽只有较高的固定荧光F。没有可变荧光Fv;原位低温荧光光谱检测表明莲胚芽只在679nm处有一个荧光发射主峰，没有正常的PSII和PSI荧光发射峰(683nm、692nm和730nm);部分变性的叶绿素蛋白复合物凝胶电泳分析结果表明莲胚芽叶绿体类囊体膜上只存在LHCII 一种叶绿素蛋白复合物（其中单体和二聚体形式的LHCII均有发现）;Western Blots检测结果表明莲胚芽的LHCII组成比较单一，同时确证了莲胚芽不含有PSI的核心和天线蛋白组分。莲胚芽LHCII和莲叶LHCII在SDS-PAGE图谱上迁移距离相同，但是光谱分析表明二者不仅在Chla、Chlb的相对含量上不同，而且在叶绿素分子与蛋白的结合状态上也存在差异，这些差异主要是由一部分Chla分子造成的，Chlb分子在二者中的结合状态则比较～致。 2，对莲胚芽成熟过程中的光合系统发育进行研究，结果表明这个过程可以分为建成期（0-20天）、稳定期（20-30天）和降解期（30—40天）三个阶段。在建成期和稳定期内，莲胚芽外面的包被物可能不是完全遮光的，所以莲胚芽能感受到环境光信号，其叶绿素合成已经光合系统建成集中在此阶段内进行：在莲’胚芽成熟后期，莲胚芽外面的包被组织开始木质化，光信号无法再穿透它们，莲胚芽的光合系统发育进入降解期，叶绿素合成停止，己建成的光合系统开始降解，到莲胚芽成熟时，除LHCIl外，光合系统其余的叶绿素蛋白复合物都被降解了，所以莲胚芽具有不同于一般祓子植物叶组织的色素和光合系统组成。对莲胚芽的成熟发育过程进行遮光处理，结果发现遮光发育的莲胚芽发生明显黄化，这表明莲胚芽的叶绿素合成也离不开光照，在莲总基因组中检测不到编码DPOR的三个基因的同源序列，确证了莲胚芽不具有在黑暗中合成叶绿素的能力。 3，在黑暗中萌发生长的莲胚芽能够在相当长的时间内保持其叶绿素稳定，特别是Chla的含量在暗生长10天以内基本没有变化;原位低温荧光光谱检测表明暗萌发过程中莲苗有PSII和PSI的荧光发射峰形成，暗生长10天左右的莲苗具有比较明显的光合系统荧光发射峰，但是与自然光照下的发育过程相比，暗萌发莲苗的光合系统荧光发射峰出现较慢，而且PSI的荧光发射相对较弱;暗萌发莲苗在转绿以及冻融过程中的原位低温荧光光谱变化表明莲苗在黑暗中建成的光合系统不完善并且不稳定;对莲胚芽、暗萌发莲苗以及莲叶的叶绿体吸收光谱进行比较，结果显示暗萌发莲苗的叶绿体发育阶段介于莲胚芽和莲叶之间;叶绿素蛋白复合物凝胶电泳分离，SDS-PAGE，Western Blots免疫检测、以及叶绿素荧光诱导动力学结果均确证暗萌发莲苗有光合系统的发育，特别是PSI的出现;对暗萌发莲苗的光化学活性进行分析，结果表明暗中建成的PSII和PSI均具有电子传递活性：但是放氧复合物的发育不完全，对莲胚芽暗萌发过程光合系统建成的原因进行分析，推测叶绿素可能起了至关重要的作用，光对于莲胚芽萌发过程中的光合系统发育来说可能并不是必需的。

17 beta-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys

BACKGROUND: Effects of 17beta-estradiol and progesterone on rhesus monkey oocyte maturation in vitro were evaluated by embryo development subsequent to IVF. METHODS AND RESULTS: In experiment 1, immature cumulus-oocyte complexes collected from unstimulated adult females during the non-breeding season were matured in modified medium CMRL-1066 containing various combinations of gonadotrophins (FSH + LH), estradiol and/or progesterone. Formation of morulae and blastocysts was greatest in oocytes matured in medium containing estradiol and/or progesterone, with or without gonadotrophins (morula 38-46%, blastocyst 14-20%) than in control oocytes matured without estradiol or progesterone (morula 14%, blastocyst 0%). In experiment 2, cumulus-oocyte complexes from unstimulated prepubertal female monkeys were matured in medium with gonadotrophins, estradiol or progesterone. The best development to the morula stage was obtained with oocytes matured with gonadotrophins and estradiol or gonadotrophins and progesterone (43 and 25 morulae, respectively), while control oocytes matured with gonadotrophins but without steroid hormones gave the poorest morula developmental response (12%). However, there was no difference in blastocyst development across all groups (0-3%). CONCLUSIONS: These results demonstrate that during rhesus monkey oocyte maturation in vitro: (i) estradiol or progesterone can improve oocyte developmental competence; (ii) immature oocytes from prepubertal versus adult females have differential responses to challenge with estradiol or progesterone.

The giant panda (Ailurapoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of interspecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the Lest, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined with in vivo development in ligated rabbit oviduct, achieved higher blastocyst development than in vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the interspecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not species-specific; (ii) there is compatibility between interspecies somatic nucleus and ooplasm during early development of the reconstructed egg.

Epigenetic marks in cloned rhesus monkey embryos: Comparison with counterparts produced in vitro

Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In man

HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo. (C) 2010 Elsevier Inc. All rights reserved.