Hira基因的过量表达对果蝇早期胚胎发育的影响

Hir／Hira基因产物是组蛋白基因表达的一种负调节因子,其在果蝇发育过程中的作用还没有得到确认．本研究将果蝇Hira基因（dHira）的cDNA克隆到载体UAsP中,运用UAS—Gal4系统使其在果蝇早期胚胎中大量表达．结果发现在胚胎发育早期,无论是在头部还是在全胚胎过量表达Hira,都引起果蝇胚胎大量死亡,而且随着转基因拷贝数的增加,胚胎的死亡率也显著增加,说明Hira过量表达对果蝇胚胎发育产生严重影响．由于Hira基因产物与核小体的组装、染色质结构等的调节有关,因此推测Hira过量表达可能是通过对组蛋白的抑制对果蝇胚胎发育造成影响的．     

非整倍体和人类生殖健康

非整倍体即染色体数目异常,发生于生殖细胞则可能导致不育不孕、自发流产和先天出生缺陷.精子非整倍体发生率为5%～7%,卵子非整倍体发生率为22%～90%;在早期自然流产中,非整倍体率高达50%.绝大多数临床上常见的非整倍体患儿的异常染色体来自卵子(母亲),而且母亲减数分裂I同源染色体不分离是除13和18号染色体以外的各种常见染色体非整倍体的主要原因.母亲的年龄是迄今唯一被证实的与生殖细胞非整倍体发生密切相关的流行病学因素,减数分裂遗传重组(频率和位点分布)异常可能是导致减数分裂I同源染色体不分离的主要细胞学和遗传学因素,减数分裂前最后一次DNA复制时cohesin复合体的加载及其之后的维持异常则可能是引起减数分裂染色体不分离的分子生物学因素.减数分裂遗传重组和cohesin复合体的加载均发生于女性胚胎发育的8～30周,而非整倍体卵子的形成多发生在35岁以上的女性,因此我们认为导致非整倍体卵子形成的"罪恶种子"早在35年前即已埋下,随着女性年龄增大,其体内保障染色体精确分离的保护机制如纺锤体聚合检验点被"磨损"削弱,而最终导致减数分裂时染色体分离异常.未来的研究应着重探讨遗传重组改变的原因、机理及如何导致减数分裂染色体不分离,了解卵母细胞中cohesin复合体、纺锤体聚合检验点的功能是否随女性年龄增大而减弱及其生物学机制,从而为有效防止非整倍体的发生、减少人类生殖相关非整倍体疾病的发生提供理论基础.     

小鼠四倍体半克隆胚胎发育研究

半克隆(Semi-Cloned)胚胎是通过注射体细胞核到未去核的卵母细胞中产生的。在半克隆胚胎中,体细胞被用来作为精子的替代物。然而,由于异常的染色体分离,构建的半克隆胚胎在激活后形成了非整倍体而导致胚胎发育受到严重影响,不能发育到期。本研究通过抑制小鼠半克隆胚胎在激活过程中染色体数目减半,避免非整倍体胚胎形成,研究四倍体半克隆(TetraploidSemi-cloned,TSC)胚胎的发育和体细胞核的掺入对胚胎发育的影响。结果显示,TSC胚胎的体外发育率显著高于二倍体半克隆胚胎,与正常受精卵及孤雌激活对照无显著性差异,但TSC胚胎的细胞数在桑椹胚和囊胚期比正常二倍体受精胚胎和孤雌激活胚胎少。通过Oct-4染色发现,TSC胚胎囊胚期内细胞团(InnerCellMass,ICM)细胞很少或者没有。移植63个四倍体半克隆胚胎到3只假孕母鼠体内,得到20个胎盘,但没有得到胎儿。组蛋白乙酰化和DNA甲基化检测显示,部分TSC胚胎在囊胚期没有形成正常受精胚胎在ICM和滋养外胚层(Trophectoderm,TE)之间的差异分布。TSC胚胎的基因表达不依赖于细胞分裂次数而依赖于发育时间。虽然TSC胚胎避免了二倍体半克隆胚胎形成非整倍体现象,但由于TSC胚胎没有ICM细胞或ICM细胞很少,所以只能形成胎盘而不能形成胎儿。本实验第一次较为全面地研究了TSC胚胎的发育,同时也为研究体细胞核再程序化、基因打靶技术提供了一种新的途径。     

AAPH诱导的氧化应激对小鼠早期胚胎发育的作用及机制研究

哺乳动物胚胎发育是一个受复杂信号通路严格调控的过程,同时受遗传和环境因素等多种因素影响.由体外培养环境破坏活性氧ROS平衡所引起的氧化应激反应被认为是影响体外胚胎发育的重要因素之一,但活性氧影响胚胎的机制仍有待研究.本研究首先观察了叠氮化合物2,2'-偶氮二(2-脒基丙烷)盐酸盐AAPH[2,2-Azobis(2-Amidinopropane)dihydrochloride]处理后对胚胎发育的影响,检测了胚胎的活性氧水平、线粒体膜电位和细胞凋亡情况,同时检测了合子基因组激活相关基因的表达变化,针对活性氧诱导剂AAPH影响小鼠早期胚胎发育的分子机制进行研究.结果表明,AAPH能够提高小鼠胚胎的活性氧水平,影响胚胎发育率,AAPH的作用呈现剂量依赖性,这种影响是通过降低线粒体的膜电位,进而促发细胞凋亡.同时,抑制合子基因组激活相关基因Zscan4d、eIF-1A、Hspa1b、H2afz的表达.本研究探讨了活性氧影响早期胚胎发育过程的机制,为哺乳动物体外受精培养体系的优化提供了依据.     

组蛋白甲基化修饰在小鼠早期胚胎发育中的建立与调控

 组蛋白修饰作为重要的表观遗传修饰，在调控胚胎基因表达、胚胎细胞的命运决定及胚胎基因组的稳定性等方面均起了很重要的作用。微量测序技术的发展使从全基因组水平上检测植入前胚胎的组蛋白修饰成为可能。综述了近年来利用该技术对小鼠早期胚胎发育过程中的组蛋白甲基化修饰研究的最新进展，总结了在胚胎基因激活及第一次细胞分化过程中组蛋白H3K4me3和H3K27me3修饰不同的建立和动态变化趋势，这些研究为探索胚胎发育和细胞分化的表观调控机制奠定了基础。     

原位杂交检测hsp70 mRNA在黑腹果蝇胚胎中的表达

利用设计合成的地高辛标记的黑腹果蝇(Drosophila melanogaster)hsp70 DNA探针,整体原位杂交检测热休克反应中,hsp70 mRNA在胚胎发育各阶段的表达情况.实验发现,在正常温度下黑腹果蝇的胚胎检测不到hsp70 mRNA存在.在热激的果蝇胚胎中,可检测到hsp70 mRNA的广泛存在.hsp70 mRNA在热激的胚胎中,除早期细胞化囊胚的极细胞外,其余细胞均可检测到hsp70 mRNA的均一分布.随着胚胎发育,hsp70 mRNA仍广泛分布在各细胞中,晚期胚胎中神经细胞hsp70 mRNA比其余细胞的表达水平高,说明神经细胞具有更高的热诱导活性,在热激反应中hsp70 mRNA的表达水平更高.     

果蝇细胞周期蛋白Cyclin D在胚胎发育不同时期的表达

细胞周期蛋白Cyclin D（CycD）是发育中重要的蛋白,作者利用原位杂交法和免疫组化法,分别对野生型的果蝇w1118以及本研究室构建的一个转基因株系cycD-lacZ进行分析,从而建立了在果蝇发育的早期胚胎发育各阶段cycDmRNA的表达模式.结果表明：Stage 5时期,cycDmRNA集中于胚胎表面新生成的细胞处.Stage10,在发育中的神经系统,前中肠和后中肠处可以检测到cycD的mRNA.Stage14,cycD基因于中肠有着强烈的表达.cycDmRNA在胚胎发育中的表达模式的确立,为后期研究CycD在胚胎发育中所起的作用奠定了基础.     

原位杂交检测pal-1 mRNA在C.elegans野生型和突变体早期胚胎中的分布

Caenorhabditis elegans是发育生物学中的重要模式生物,许多基因参与C.elegans的细胞命运决定和细胞谱系发育.par基因的活性影响C.elegans胚胎前后轴的极性,其功能缺失会使胚胎的第一次分裂丧失前后轴的不对称性,导致一些母体提供的发育调控因子不能在特定的胚胎细胞中准确定位,从而改变胚胎细胞发育命运.pal-1是C.elegans早期胚胎发育中决定细胞命运的重要基因,它决定体细胞的性质,也是转录因子,调控着后续基因的表达,凡含有该基因表达的细胞发育成体细胞.调控PAL-1表达的基因有许多,本文通过原位杂交检测pal-1 mRNA在C.elegans野生型和par-5、par-6、gld-1突变体早期胚胎中的分布,来探讨par-5、par-6和gld-1基因的活性在胚胎发育早期对pal-1 mRNA的影响.实验结果表明,pal-1mRNA在野生型早期胚胎中有明显极性,而pal-1mRNA分布的不对称性在上述基因缺失的情况下被破坏,这些基因与pal-1mRNA的不对称分布直接相关.     

果蝇细胞周期蛋白Cyclin D在胚胎发育不同时期的表达

细胞周期蛋白Cyclin D(CycD)是发育中重要的蛋白,作者利用原位杂交法和免疫组化法,分别对野生型的果蝇w1118以及本研究室构建的一个转基因株系cycD-lacZ进行分析,从而建立了在果蝇发育的早期胚胎发育各阶段cycD mRNA的表达模式.结果表明:Stage 5时期,cycD mRNA集中于胚胎表面新生成的细胞处.Stage10,在发育中的神经系统,前中肠和后中肠处可以检测到cycD的mRNA.Stage14,cycD基因于中肠有着强烈的表达.cycD mRNA在胚胎发育中的表达模式的确立,为后期研究CycD在胚胎发育中所起的作用奠定了基础.     

p21基因在小鼠胚胎发育过程中的表达

以E9日龄至E14日龄昆明种正常小鼠胚胎为材料,利用质粒扩增的、地高辛标记的基因探针在组织切片上进行DNA-mRNA分子原位杂交,研究了p21基因在小鼠胚胎发育过程中的表达.结果表明:p21基因从E10日开始参与小鼠胚胎发育,其表达特异性随着胚胎发育进程逐渐增强,与它在细胞周期中的负调控作用相一致.p21基因表达强度较稳定,与其mRNA稳定有关.     

Embryological and molecular investigations of parental imprinting on mouse chromosome 7

Mouse embryos with duplications of whole maternal (parthenogenetic and gynogenetic) or paternal (androgenetic) genomes show reciprocal phenotypes and do not develop to term. Genetic complementation has identified the distal region of chromosome 7 (Chr 7) as one of the regions for which both a maternal and paternal chromosome copy are essential for normal development, presumably because of the presence of imprinted genes whose expression is dependent on their parental origin. Embryos with the maternal duplication and paternal deficiency of distal Chr 7 are growth retarded and die around day 16 of gestation; the reciprocal paternal duplication embryos die at an unidentified earlier stage. We report here the incorporation of cells with the paternal duplication into chimaeras, resulting in a striking growth enhancement of the embryos. One gene located on mouse distal Chr 7 (ref. 5) is the insulin-like growth factor 2 (Igf2) gene, an embryonic mitogen. In embryos with the maternal duplication of distal Chr 7, the two maternal alleles of the Igf2 gene are repressed. The presence of two paternal alleles of this gene in many cells is probably responsible for the growth enhancement observed in chimaeras. We propose that there are other imprinted genes in this Chr 7 region. We also compare the imprinting of this subgenomic region with phenotypes resulting from the duplication of the whole parental genome in parthenogenones and androgenones.     

Regulation of the Drosophila segmentation gene hunchback by two maternal morphogenetic centres

Segmentation in the inset embryo is initiated by maternally provided information, which is stored in the developing oocyte. In Drosophila, the genes necessary for this process have been genetically characterized. The anterior segmented region is organized by the bicoid (bcd) gene product. The posterior segmented region is organized by several interacting gene products, among them the oskar (osk) gene product. The first zygotic group of genes, which are thought to respond to the spatial cues provided by the maternal genes, are the gap genes, whose members include hunchback (hb), Krüppel (Kr) and knirps (kni). To elucidate the role played by the maternal genes in expression of the gap gene hb, antibodies were raised against a fusion protein and were used for the cytological localization of the hb gene product in wild-type and mutant embryos. The hb protein is predominantly located in the nucleus. Its spatial expression includes the formation of an anterior-posterior gradient during the early cleavage stages and a strong zygotic expression in the anterior half of the embryo. Analysis of embryos mutant for the maternal genes affecting the anterior-posterior segmentation pattern shows that the formation of the early gradient is controlled by the osk group of genes, whereas efficient activation of the zygotic anterior expression domain is dependent on bcd activity.     

Cloning and roles of goldfish maternal factor β-Catenin cDNA in embryonic development

Interaction between nucleus and cytoplasm has been focused in the field of animal embryonic development, in which study of maternal factors is required positively. β-Catenin, an important maternal factor in early embryogenesis, has been analyzed in its expression pattern and functions in this paper. We have cloned goldfish β-Catenin cDNA gene and compared it with zebrafish β-Catenin cDNA. High homology was found in cDNA and in amino acid sequences between them, 93% (2227/2384 bp) and 98.5% (768/780 aa) respectively. The expression pattern of β-Catenin by in situ hybridization and the roles of β-Catenin on embryonic development by co-injection of anti-sense RNA and reporter gene, EGFP have been investigated in the whole process of goldfish embryonic development. The results suggest that β-Catenin presents dynamic distribution, mainly locates at body axis, dorsal tissues, head and tail structures after being fertilized. The loss of β-Catenin activity would cause serious destruction of embryo in dorsal tissues and in anteroposterior axes, and leads embryos to die before larva get hatched.     

The Drosophila posterior-group gene nanos functions by repressing hunchback activity

The development of the body plan in the Drosophila embryo depends on the activity of maternal determinants localized at the anterior and posterior of the egg. These activities define both the polarity of the anterior-posterior (AP) axis and the spatial domains of expression of the zygotic gap genes, which in turn control the subsequent steps in segmentation. The nature and mode of action of one anterior determinant, the bicoid(bcd) gene product, has recently been defined, but the posterior determinants are less well characterized. At least seven maternally acting genes are required for posterior development. Mutations in these maternal posterior-group genes result in embryos lacking all abdominal segments. Cytoplasmic transplantation studies indicate that the maternally encoded product of the nanos(nos) gene may act as an abdominal determinant, whereas the other maternal posterior-group genes appear to be required for the appropriate localization and stabilization of this signal. Here we show that the lack of the nos gene product can be compensated for by eliminating the maternal activity of the gap gene hunchback (hb). Embryos lacking both of these maternally derived gene products are viable and can survive as fertile adults. These results suggest that the nos gene product functions by repressing the activity of the maternal hb products in the posterior of the egg.     

DAP 玻璃化冷冻液对小鼠2-细胞胚胎的冷冻保存

摘要: 目的探索和优化小鼠2-细胞胚胎冷冻效果,建立小鼠胚胎冷冻保存技术以及超低温保存模型动物胚胎实验室所需的相关技术。方法选择3 个品系( C57BL/6、ApoE － / －、NOS3 － / － ) SPF 级4 周龄雌性小鼠,运用超数排卵、体外受精、玻璃化冷冻法,以及二细胞期胚胎复苏、体外培养等技术方法,观察上述实验效果。结果C57BL/6、ApoE － / －、NOS3 － / － 3 个品系小鼠超数排卵平均值分别为42. 98 个/只、27. 93 个/只、23. 11 个/只,体外受精2-细胞期胚胎率分别为68. 79%、32. 70%、23. 08 %,胚胎冷冻复苏后胚胎回收率72. 77%、80. 87%、83. 33%,存活率分别为56. 92%、54. 84%、70%,存活胚胎体外培养发育到囊胚比率分别为72. 37%、60. 78%、25%。结论选择4 周龄小鼠,按预定相同的技术方法做超数排卵、体外受精、胚胎冻存、胚胎复苏,呈现出较好效果,C57BL/6 小鼠优于基因工程小鼠,基本建立起小鼠2-细胞胚胎冷冻保存技术。     

Derivation of androgenetic embryonic stem cells from m-carboxycinnamic acid bishydroxamide (CBHA) treated androgenetic embryos

The androgenetic embyronic stem (aES) cells are useful models in studying the effects of imprinted genes on pluripotency maintaining and embryo development. The expression patterns of imprinted genes are significantly different between uniparental derived aES cells and zygote-derived embryonic stem (ES) cells, therefore, the imprinting related cell pluripotency needs further exploitation. Several approaches have been applied in generation of androgenetic embryos and derivation of aES cell lines. Here, we describe a method to generate androgenetic embryos by injecting two mature sperms into one enucleated oocyte. Then these androgenetic embryos were treated with a histone deacetylase inhibitor: m-carboxycinnamic acid bishydroxamide (CBHA). Further, aES cell lines were successfully derived from these treated androgenetic embryos at blastocyst stage. The CBHA could improve not only the quality of androgenetic embryos, but also the efficiencies of aES (CaES) cells derivation and chimeric mice generation. The imprinted gene expression pattern in the CBHA treated embryo-derived aES (CaES) cells was also highly similar to that of zygote-derived ES cells.