Reconstitution of Gametogenesis In Vitro:Meiosis Is the Biggest Obstacle

Germ-line cells are responsible for transmitting genetic and epigenetic information across generations, and ensuring the creation of new individuals from one generation to the next. Gametogenesis process requires several rigorous steps, including primordial germ cell （PGC） specification, proliferation, migration to the gonadal ridges and differentiation into mature gametes such as sperms and oocytes. But this process is not clearly explored because a small number of PGCs are deeply embedded in the developing embryo. In the attempt to establish an in vitro model for understanding gametogenesis process well, several groups have made considerable progress in differen- tiating embryonic stem cells （ESCs） and adult stem cells （ASCs） into germ-like cells over the past ten years. These stem cell-derived germ cells appear to he capable of undergoing meiosis and generating both male and female gametes. But most of gametes turn out to be not fully functional due to their abnormal meiosis process compared to endogenous germ cells. Therefore, a robust system of differentiating stem cells into germ cells would enable us to investigate the genetic, epigenetic and environmental factors associated with germ cell development. Here, we review the stem cell-derived germ cell development, and discuss the potential and challenges in the differentiation of functional germ cells from stem cells.     

β-amylase in developing apple fruits: activities, amounts and subcellular localization

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. Based on previously reported in vitro assays, β-amylase is considered one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was shown often extrachloroplastic in living cells. The present experiment showed that β-amylase activity was progressively increasing concomitantly with decreasing starch concentrations during apple (Malus domestica Borkh cv. Starkrimson) fruit development. The apparent amount of β-amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The subcellular-localization studies via immunogold electron-microscopy technique showed that β-amylase visualized by gold particles was predominantly located in plastids especially at periphery of starch granules, but the gold particles were scarcely found in other subcellular compartments. These data proved for the first time that the enzyme is compartmented in its functional sites in plant living cells. The predominantly plastid-distributed pattern of β-amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (β-amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that β-amylase is involved in starch hydrolysis in plastids of the fruit cells.     

Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes,followed by major epigenetic remodeling leading to the formation of a totipotent embryo.During the development of the pre-implantation embryo,precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality,but the underlying molecular mechanisms remain elusive.For the past few years,unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development,taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies.The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals,including DNA methylation,histone modifications,chromatin accessibility and 3D chromatin organization.     

Retarded Embryo Development 1(RED1) regulates embryo development,seed maturation and plant growth in Arabidopsis

Plant seeds accumulate large amounts of protein and carbohydrate as storage reserves during maturation.Thus,understanding the genetic control of embryo and seed development may provide bioengineering tools for yield improvement.In this study,we report the identification of Retarded Embryo Development 1(RED1) gene in Arabidopsis,whose two independent T-DNA insertion mutant lines,SALK_085642(red1-1) and SALK_022583(red1-2),show a retarded embryo development phenotype.The embryogenesis process ceases at the late heart stage in red 1-1 and at the bent-cotyledon stage in red 1-2,respectively,resulting in seed abortion in both lines.The retarded embryo development and seed abortion phenotypes reverted to normal when RED1 complementation constructs were introduced into mutant plants.Small red 1-2 homozygous plants can be successfully rescued by culturing immature seeds,indicating that seed abortion likely results from compromised tolerance to the desiccation process associated with seed maturation.Consistent with this observation,red 1-2 seeds accumulate less protein,and the expression of two late embryo development reporter transgenes,LEA::GUS and β-conglycinin::GUS,was significantly weak and started relatively late in the red 1-2 mutant lines compared to the wild type.The RED1 gene encodes a plant specific novel protein that is localized in the nucleus.These results indicate that RED1 plays important roles in embryo development,seed maturation and plant growth.     

Recent advances in bone regeneration using adult stem cells

Bone is a highly vascularized tissue reliant on the close spatial and temporal association between bloodvessels and bone cells. Therefore, cells that participate in vasculogenesis and osteogenesis play a pivotal role in bone formation during prenatal and postnatal periods. Nevertheless, spontaneous healing of bone fracture is occasionally impaired due to insufficient blood and cellular supply to the site of injury. In these cases, bone regeneration process is interrupted, which might result in delayed union or even nonunion of the fracture. Nonunion fracture is difficult to treat and have a high financial impact. In the last decade, numerous technological advancements in bone tissue engineering and cell-therapy opened new horizon in the field of bone regeneration. This review starts with presentation of the biological processes involved in bone development, bone remodeling, fracture healing process and the microenvironment at bone healing sites. Then, we discuss the rationale for using adult stem cells and listed the characteristics of the available cells for bone regeneration. The mechanism of action and epigenetic regulations for osteogenic differentiation are also described. Finally, we review the literature for translational and clinical trials that investigated the use of adult stem cells(mesenchymal stem cells, endothelial progenitor cells and CD34+ blood progenitors) for bone regeneration.     

ABA-Mediated Inhibition of Germination Is Related to the Inhibition of Genes Encoding Cell-Wall Biosynthetic and Architecture： Modifying Enzymes and Structural Proteins in Medicago truncatula Embryo Axis

Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid （ABA） but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.     

Simulation of Fluid Flow in a Channel Induced by Three Types of Fin-Like Motion

One of many interesting research activities in biofluidmechanics is dedicated to investigations of locomotion in water. Some of propulsion mechanisms observed in the underwater world are used in the development process of underwater autonomic vehicles （AUV）. In order to characterise several solutions according to their manoeuvrability, influence on the surrounding fluid and energetic efficiency, a detailed analysis of fin-like movement is indispensable. In the current paper an analysis of undulatory, oscillatory and combined fin-like movements by means of numerical simulation is carried out. The conservation equation of mass and the conservation equation of momentum axe solved with the Finite Volume Method （FWM） by use of the software CFX-10.0. The undulatory and oscillatory fin movements axe modelled with an equation that is implemented within an additional subroutine and joined with the main solver. N carried out in the computational domain, in which one fin is fixed in a flow-through water duct. Simulations axe carded out in the range of the Re number up to 105. The results show significant influence of applied fin motion on the velocity distribution in the surrounding fluid.     

The giant panda (Ailuropoda 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 best, 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 blastoeyst 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 (     

肾蕨配子体发育的研究

利用光学显微镜观察肾蕨(Nephrolepis auriculata)孢子的萌发及配子体发育过程。结果表明:成熟的孢子棕黄色,不透明,极面观圆球形,赤道面观豆形,单裂缝,表面光滑。孢子萌发类型为书带蕨型,播种后15 d左右萌发,形成5-12个细胞长的丝状体。原叶体发育为三叉蕨型。30 d左右发育为片状体,45 d左右形成幼原叶体,幼原叶体不对称,成熟原叶体心脏形对称。原叶体边缘及背腹面都具毛状体,毛状体由单细胞构成。60 d左右精子器开始出现,精子器近圆球形,由3个细胞构成。70 d左右颈卵器出现,成熟颈卵器颈部由4列细胞构成,3-5层细胞高。原叶体受精后1个月内可看到幼胚生成。     

粗齿黔蕨配子体发育的研究

用原生境腐殖土对粗齿黔蕨(Phanerophlebiopsis blinii(Lévl.)Ching)的孢子进行培养,显微镜下观察孢子萌发和配子体发育过程.结果表明:粗齿黔蕨的孢子深棕色,单裂缝,极面观椭圆形,赤道面观为半圆形.播种1周后孢子萌发,萌发类型为书带蕨型(Vittaria-type),配子体发育为叉蕨型(Aspidium-type).丝状体由4～12个细胞组成,片状体宽达9个细胞,斜向一侧生长,边缘具毛状体.播种约2个月后形成原叶体,成熟原叶体成对称心形.在粗齿黔蕨的配子体发育过程中,成熟原叶体的背腹面和边缘均被毛状体,假根有分叉且尖部常膨大,并含有较大的颗粒状贮藏物,精子器有3个壁细胞等特征较为进化,而颈卵器粗短且直立的特征较为原始.粗齿黔蕨的精子器和颈卵器发育不同步,精子器的出现和成熟均早于颈卵器的发育.从配子体发育的角度,初步探讨了粗齿黔蕨野外种群数量较少的成因.     

竹叶蕨配子体发育的培养观察

首次在光学显微镜下观察竹叶蕨孢子及其萌发、丝状体发育、片状体和生长点的形成及分化、原叶体细胞形态、假根及性器的发育等方面所表现出的显微特征。初步讨论竹叶蕨科从鳞始蕨科中分立出来的合理性,以及原叶体边缘细胞的形态、叶绿体对光的敏感性、假根的形态和精子器的形成及分化的系统学意义。     

水蕨配子体发育的研究

研究了我国产水蕨(Ceratopteris thalictroides)配子体发育的全过程,水蕨孢子为四面体型,三裂缝,孢子萌发为向心型,丝状体4～8个细胞长,无明显的顶端细胞,丝状体顶部细胞同时分裂形成片状体,分生组织位于片状体一侧,使原叶体成为不对称的心脏形,原叶体发育为水蕨型,无毛状体,精子器和颈卵器较大,该配子体表现为较原始的性状.     

药用植物乌蕨配子体发育的观察

药用植物乌蕨(Stenoloma chusanum(L.)Ching)配子体发育周期短而迅速,是一种研究蕨类配子体世代的生理生态和分子生物学等特征的理想试验材料.本文采用原生境腐殖土培养方法对乌蕨的孢子进行人工培养,观察并记录其孢子萌发和配子体发育的过程.结果表明:乌蕨孢子呈黄色,单裂缝,二面体形;孢子萌发为书带蕨型(Vittaria-type).丝状体4～6个细胞,有时具二叉分枝.片状体为长楔形,达6～8个细胞宽.成熟的原叶体为对称心形,裸露,发育为槲蕨型(Drynaria-type),并且具备一定的营养繁殖能力.原叶体上的假根为单细胞且不含叶绿体.精子器为盖裂开放,颈卵器短而直立.利用孢子培养的方法对乌蕨种苗进行人工繁殖,将为保护性开发利用这一重要药用资源奠定基础.     

桫椤科三种植物配子体发育的研究

用土壤培养桫椤Alsophila spinulosa(Wall.exHook.)R.M.Tryon、中华桫椤A.costularis Baker和白桫椤Sphaeropteris brunoniana(Hook.)R.M.Tryon的孢子,利用光学显微镜对其配子体发育的各个阶段进行了观察,包括孢子形态及其萌发、原丝体发育特点、片状体和生长点的形成及分化、假根特征、精子器和颈卵器的分化及发育,以及原叶体感染真菌后的显微特征。初步讨论了桫椤科不同属(种)间的配子体发育的系统学意义和有性世代的濒危原因。     

香鳞毛蕨配子体发育的研究

研究了香鳞毛蕨(Dryopteris fragrans (L.)Schott)配子体的发育过程。结果发现孢子极面观为椭圆形,赤道面观为半圆形,单裂缝。孢子萌发为书带蕨型;丝状体2～9个细胞长,有明显顶端细胞,可形成多细胞的广阔板状片状体;片状体顶端分生组织继续分裂,发育为幼原叶体;原叶体发育为三叉蕨型;毛状体数量丰富,均为单细胞;颈卵器和精子器几乎同时形成,较小;成熟原叶体倒卵状心脏形。该配子体表现为进化性状。     

下延叉蕨和芽胞叉蕨配子体发育的研究

利用光学显微镜详细观察了叉蕨属(Tectaria)下延叉蕨(Tectaria decurrens)和芽胞叉蕨(T.fauriei)的配子体发育过程,记录了配子体各发育阶段的特征。结果表明:(1)下延叉蕨和芽胞叉蕨的孢子均为单裂缝,具周壁,由周壁形成纹饰,孢子极面观椭圆形,赤道面观豆形或肾形。(2)孢子萌发方式为向心型。(3)原叶体发育方式为三叉蕨型。(4)成熟原叶体心脏形,两翼向斜上方扩展。(5)均具单细胞和多细胞毛状体,在丝状体或片状体阶段出现。研究认为,从配子体发育角度看,叉蕨属是较进化的陆生真蕨类;毛状体的类型、位置和出现时间等特征在叉蕨属种间存在差异,可作为该属种间分类的特征。     

苏铁蕨配子体发育的研究

用无机培养基培养苏铁蕨(Brainea insignis (Hook.)J.Sm.)的孢子,显微镜下观察记录其孢子萌发及配子体形态发育过程.结果表明:孢子褐色,单裂缝,具周壁,稍褶皱.接种3 d左右孢子萌发,萌发类型为书带蕨型(VittariaType),原叶体发育类型为槲蕨型(Drynaria Type).接种15 d左右发育为片状体.接种25 d左右发育为成熟原叶体,呈心形,其翼面和翼缘均分布有毛状体.精子器由3细胞构成,成熟颈卵器颈部由4列细胞组成,4～5层细胞高.     

四回毛枝蕨配子体发育的研究

采用MS培养基培养四回毛枝蕨孢子,利用光学显微镜详细观察记录了其孢子萌发、配子体发育及幼孢子体形成的整个过程。结果表明:成熟的孢子黑褐色,不透明,极面观圆球形,赤道面观蚕豆型,单裂缝,表面微褶皱。播种后8d左右萌发,萌发类型为书带蕨型,配子体发育为三叉蕨型。播种20d左右发育为片状体。播种30d左右形成幼原叶体,幼原叶体暂不对称,成熟原叶体呈蝴蝶形。原叶体边缘及背腹面都具毛状体,数量丰富,单细胞。播种50d左右开始有性器官出现,精子器近圆球形,由3细胞构成,成熟颈卵器颈部由5列细胞构成。原叶体受精后1月内可看到幼胚生成。