Orientation of microtubules suggests a role in mRNA transportation in fertilized eggs of Chinese pine (Pinus tabulaeformis)

Summary. Polysomes become associated with microtubules (MTs) in egg cells of Chinese pine upon fertilization, providing direct evidence for MT-based intracellular mRNA and polysome localization. We have investigated by immunoelectron microscopy the orientation and spatial distribution of MTs and their association with polysomes in the fertilized egg cells. There is a perinuclear accumulation of MTs and polysomes in the zygote soon after fertilization. At this time, some of the MTs are perpendicular to the nuclear envelope and directly connected to the outer membrane or nuclear-pore complexes (NPC) at one end, and the other ends reach to the outer tier or cortical MTs that are parallel to the long axis of the zygote. The polysomes in the perinuclear region show the same spatial and temporal pattern as the MTs. Immunolocalization of the mRNA-binding protein hnRNP indicates that the mRNAs are loaded onto the nucleus-associated MTs immediately after their export from the nuclear-pore complexes. The polysomes and mRNAs are then transported from these MTs to the outer tier and/or cortical MTs, where they further localize to the polar region of the cell. Correspondence (present address): Fengli Guo, Stowers Institute for Medical Research, 1000 E 50th Street, Kansas City, MO 64110, U.S.A.     

Actin gene expression in developing sea urchin embryos.

We show that the synthesis of actin is regulated developmentally during early sea urchin embryogenesis and that the level of synthesis of this protein parallels the steady-state amounts of the actin messenger ribonucleic acids (RNA). An in vitro translation and RNA blotting analysis of embryo RNA from several stages of early development indicated that during the first 8 h after fertilization there was a low and relatively constant level of actin messenger RNA in the embryo. Between 8 and 13 h of development, the amount of actin messenger RNA began to increase both in the cytoplasm and on polysomes, and by 18 h the amounts of actin message per embryo had risen between approximately 10- and 25-fold in the cytoplasm and between 15- and 40-fold on polysomes. Two size classes of actin messenger RNA (2.2 and 1.8 kilobases) were identified in unfertilized eggs and in all of the developmental stages examined. The amount of each actin message class increased over a similar time interval during early development. However, the amounts of these size classes in the cytoplasm relative to each other shifted between the earliest stages examined (2 to 5 h) and the hatching blastula stage (18 h), with the ratio of the 1.8-kilobase actin messenger RNA to the 2.2-kilobase actin messenger RNA increasing almost threefold during this period.     

CAPSELLA EMBRYOGENESIS: THE EGG,ZYGOTE, AND YOUNG EMBRYO

The ultrastructure and composition of the egg, zygote, and young embryo of Capsella bursa-pastoris were examined. The egg is a highly polarized cell; one-half to one-third of the micropylar end is filled with a large vacuole while the chalazal end contains the nucleus and much of the cytoplasm of the cell. The wall which surrounds the cell is incomplete at the chalazal end. Ribosomes fill the cytoplasm and show little or no aggregation into polysomes. The structure of the nucleolus suggests that ribosomes are not being produced. Following fertilization and the formation of the zygote, the cell decreases slightly in volume as the large central vacuole becomes smaller. The zygote soon increases in size as the small chalazal vacuoles present before fertilization begin to enlarge. The dictyosomes become active and a continuous wall forms around the zygote. Aggregation of the ribosomes begins and numerous polysomes are formed. Before division of the zygote all plasmodesmata between the zygote and the surrounding cells are lost. The first division of the zygote is unequal as a result of its marked polarity. A large basal cell and a small terminal cell are produced. The basal cell appears to contain more protein, RNA, carbohydrate, and cell organelles than the terminal cell. Ribosomal aggregation is even more pronounced at this stage. Starch accumulates in the plastids. Numerous plasmodesmata are present between the terminal and basal cells but there are no connections between the endosperm or other cells. The basal cell divides next to give rise to a three-celled linear embryo consisting of the basal cell, the suspensor cell, and the terminal cell. The terminal cell stains more intensely for protein and RNA as a result of increased numbers of ribosomes. Starch in all the cells is about equal and reaches a maximum in the embryo at this stage.     

The Ultracytochemical Localzation of Acid Phosphatase Activity in Wheat During Fertilization

No acid phosphatase activity was observed in the mature embryo sac of wheat (Triticum aestivum) except the chalazal cytoplasm Of the central cell before fertilization. During fertilization, acid phosphataseactivity was observed in the following loci: part of chromatin of the egg nucleus and most of the mitochondria in the egg cytoplasm; the perinuclear spaces of the egg and sperm nuclei at the fusion of the egg and sperm nuclei; the chalazal cytoplasm and some vacuoles of the degenerated synergid; two sperm nuclei within the cytoplasm of female cells; the cell wall of each cell of the embryo sac and that of the nucellar cells surrounding the embryo sac. No acid phosphatase was observed in the two-celled proembryo. Dense enzyme reaction product was localized in the chromatin of the free nuclei at early stage of the endosperm. The characteristic of acid phosphatase distribution during fertilization may be associated with the physiological change of the egg Cell, the reorganization of mitochondria in the egg cell cytoplasm, the degeneration of one of the two synergids, the physiological state of the sperm nuclei and the nuclear membrane fusion of the egg and sperm nuclei.     

小麦受精过程中酸性磷酸酶的超微细胞化学定位

小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）受精前成熟胚囊,除胚囊中央细胞的合点端细胞质中有酸性磷酸酶外,其余部位均未发现酸性磷酸酶。受精时期,以下部位存在酸性磷酸酶活性：卵细胞的细胞核内一部分染色质和细胞质中大部分线粒体;精、卵核融合时两核的核周腔内;退化助细胞合点端细胞质和一些液泡内;进入雌性细胞中的两个精核;胚囊各成员细胞的细胞壁及胚囊周围珠心细胞的细胞壁。二细胞原胚中未见有酸性磷酸酶。早期胚乳游离核染色质上有酸性磷酸酶。小麦受精过程酸性磷酸酶的分布特点可能与卵细胞生理状态的变化和细胞质中线粒体的改组、助细胞的退化、精核的生理状态以及精核与卵核的核膜融合等有关。     

Transition of Xwnt-11 mRNA from inactive form to polyribosomes in frogs during early embryogenesis

The distribution of Xwnt-11 mRNA between polysomes and informosomes was studied in Xenopus laevis and Rana temporaria during early embryogenesis. The ratio between polysomes and informosomes suggests their involvement in translation of these mRNAs. In eggs and immediately after fertilization the Xwnt-11 mRNAs are mostly positioned in informosomes. During the cleavage stage, these mRNAs have also been recognized in polysomes. Just before the onset of zygote genome functioning (at the stage of mid blastula), Xwnt-11 mRNA rapidly appears in polysomes of Rana embryos. However, in Xenopus, Xwnt-11 mRNA appears in polysomes only at the end of gastrula. Before this stage, the Xwnt-11 mRNA in Xenopus can be found mostly in informosomes.     

Polo-like kinase-1 in porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that regulate multiple stages of mitosis. Expression and distribution of polo-like kinase 1 (Plk1) were characterized during porcine oocyte maturation, fertilization and early embryo development in vitro, as well as after microtubule polymerization modulation. The quantity of Plk1 protein remained stable during meiotic maturation. Plk1 accumulated in the germinal vesicles (GV) in GV stage oocytes. After germinal vesicle breakdown (GVBD), Plk1 was localized to the spindle poles at metaphase I (MI) stage, and then translocated to the middle region of the spindle at anaphase-telophase I. Plk1 was also localized in MII spindle poles and on the spindle fibers and on the middle region of anaphase-telophase II spindles. Plk1 was not found in the spindle region when colchicine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. After fertilization, Plk1 concentrated around the female and male pronuclei. During early embryo development, Plk1 was found to be in association with the mitotic spindle at metaphase, but distributed diffusely in the cytoplasm at interphase. Our results suggest that Plk1 is a pivotal regulator of microtubule organization and cytokinesis during porcine oocyte meiotic maturation, fertilization, and early embryo cleavage in pig oocytes.     

Barriers to interspecific hybridization between Vigna unguiculata and Vigna vexillata

Summary Interspecific hybridization between Vigna unguiculata and V. vexillata always failed: no seed was obtained in both crossing directions. Two different barriers to crossability were found: a pre-zygotic barrier and a post-zygotic one. Many abnormalities were observed in pollen-tube development, which reduced the percentage of fertilization to 18–30%. Differences in the percentage of fertilization were detected between the two accessions of V. vexillata involved in the interspecific crosses. The development of the interspecific embryo was analyzed and the embryo and endosperm nuclei always degenerated 5–8 days after pollination. The growth of the embryo stopped at a globular stage, which is too early for excision and in vitro culturing.     

The microtubular cytoskeleton during development of the zygote,proembryo and free-nuclear endosperm inArabidopsis thaliana (L.) Heynh

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm inA. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.Abbreviations DIC differential interference contrast optics - MT microtubule - PPB preprophase band of microtubule We thank Ms. Margaret Travers for her helpful English translation of Yakovlev and Alimova (1976) and Mr. James Whitehead for preparation of Fig. 11. M.C.W. was supported by an Australian Postgraduate Research Award.     

Embryo sac development in Arabidopsis thaliana II. The cytoskeleton during megagametogenesis

The microtubular and actin cytoskeletons have been investigated during megagametogenesis in Arabidopsis thaliana using immunofluorescence labelling of isolated coenocytic and mature embryo sacs. We found both actin and microtubules (MTs) to occur in abundance throughout megagametogenesis and in all constituent cells of the mature embryo sac. During many stages, the patterns of distribution of these cytoskeletal elements are congruent and may prove to be co-aligned. Many changes in the arrays of MTs and microfilaments take place and indicate varying roles of the cytoskeleton in the different stages and cell types of megagametogenesis. Two major populations of MTs recur throughout embryo sac formation: (1) Elaborate nuclear-based networks are found during the two-nucleate and four-nucleate developmental stages as well as in the egg cell. These arrays may function in positioning the nuclei. (2) Cytoplasmic MTs in longitudinal orientation in the two-nucleate embryo sac, synergids and part of the egg cell, or in a reticulate pattern in the four-nucleate embryo sac, egg and central cell probably participate in organization of the cytoplasm. Synergid MTs converge at the filiform apparatus. Preprophase bands of MTs are absent throughout megagametogenesis but phragmoplast arrays occur during cellularization of the embryo sac. Well developed arrays of cortical MTs are restricted to the antipodal cells. A large concentration of MTs in the part of the egg cell adjacent to the synergids is well placed for being involved with sperm cell movement within the degenerative synergid. On the basis of the morphology of the cytoskeleton, we concur with views that the shape of megagametophyte is largely determined by the surrounding tissues, including the integumentary tapetum.     

小盐芥受精作用和胚发育的解剖学研究

用石蜡切片法对小盐芥(Thellungiella halophila(C.A.Mey.)O.E.Schulz)的受精作用及胚和胚乳的发育过程进行了解剖学观察,结果显示:花盛开前后,是受精作用的核融合时期;受精前,2个极核融合成次生核;受精作用属有丝分裂前型;胚乳发育为核型;胚发育属柳叶菜型,子叶背倚胚根;成熟种子无胚乳.     

Apomixis in plants: structural and functional aspects of diplospory inPoa nemoralis andP. palustris

Summary Data on structural and functional aspects of mitotic diplospory and later stages of apomictic seed formation are reported forPoa palustris andP. nemoralis. In this study, the plant material of two Russian populations ofP. nemoralis andP. palustris were used for transmission electron microscope observations. Seed formation was investigated by phase contrast microscopy in two populations ofP. nemoralis collected in The Netherlands. The processes of transformation of the megasporocytes to the megaspore mother cells of diplosporous embryo sacs, and thereafter to one- and two-nucleate diplosporous embryo sacs (Antennaria type) were characterized by an increase of cell size, structural and functional reorganization of the nucleus, nucleolus, and cytoplasm, and cell isolation as a result of thickening of the cell wall. These were accompanied by an increase in the cell metabolic activity inferred from visual evidence of the activation of nucleus, nucleolus, endoplasmic reticulum, dictyosomes, mitochondria, and from the appearance of a dense population of ribosomes and polysomes. The diplosporous embryo sac of the Antennaria type was characteristic for bothP. nemoralis andP. palustris. No signs of the presence of synaptonemal complexes were observed during the process of diplosporous-embryo-sac megaspore mother cell differentiation and division. About 90–95% of the diploid egg cells of diplosporous embryo sacs were able to produce apomictic embryos. These embryos developed before anthesis. However, many of them degenerated at the globular stage because of lack of endosperm. The ultrastructural events occurring during the process of diplospory of apomictic species, and meiosis and megagametogenesis of sexually reproduced plants are discussed.Abbreviations DMC megaspore mother cell of diplosporous embryo sac - TEM transmission electron microscopy - ER endoplasmic reticulum     

Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos

Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.     

Cortical ultrastructure of freeze-substituted protonemata of the mossFunaria hygrometrica

Summary The ultrastructural organization of the cortical cytoplasm has been examined in caulonemata, branches and buds of the mossFunaria hygrometrica, which were prepared by rapid freeze-fixation and freeze-substitution (FS). The same structural components occur in the cortex of all three cell types: microtubules (MTs), endoplasmic reticulum (ER), coated and uncoated vesicles, coated pits, and dictyosomes. However, the configuration and density of the cortical ER varies between the three. Caulonemata have an open, polygonal network of ER associated with long MTs oriented mostly parallel to the length of the cell. Lamellar ER, covered with polysomes, is interspersed in the network. Branches have a more tightly arranged ER network, at places occurring in a thick layer, and occasional polysome-decorated lamellae. MTs, which extend to the tip of the branch, are oriented mainly parallel to the cell's long axis and are associated with the cortical ER. Buds have the tightest ER network, which is frequently arranged in a thick layer. Tubules in the polygonal ER of buds are densely covered with ribosomes, whereas tubules in the ER network of caulonemata and branches range from nearly smooth to moderately rough. Closely-spaced ER lamellae, with many polysomes, occur in some buds. The MTs of buds extend into the apical dome and are associated with the cortical ER, but are more randomly oriented than in caulonemata or branches. Close appositions between the ER and PM are observed in all three cells, but are more frequent in buds.Abbreviations DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - FS freeze-substitution - MT microtubule - MF microfilament - PM plasma membrane     

濒危植物马蹄参的大孢子发生和雌配子体发育

为探讨马蹄参(Diplopanax stachyanthus Hand.-Mazz.)濒危机制与雌性生殖发育的关系,采用石蜡切片法观察马蹄参大孢子形成和雌配子体的发育过程。结果表明,马蹄参雌蕊单心皮,子房下位,1室,1枚胚珠。胚珠横生于短片状胎座上,具单珠被,厚珠心。单孢原细胞自珠心1~2层表皮细胞处分化;大孢子四分体为直线形。成熟胚囊中,2个极核在受精前融合为次生核,3个反足细胞不发达,较早退化;二核胚囊时期出现二核分裂不均且较小核退化消失的异常发育现象。因此,马蹄参雌配子体发育过程中出现异常现象是造成其结实率低的主要原因。     

Structure and expression of the nerve growth factor gene in Xenopus oocytes and embryos

A large part of the coding portion of the Xenopus nerve growth factor (NGF) gene has been identified and cloned by the use of a chicken cDNA probe and its sequence has been determined. Comparison of the derived amino acid sequence of mature Xenopus NGF with that of other species showed a high conservation, whereas comparison of the prepropeptide showed large divergent regions alternated with short conserved regions. Expression of the NGF gene was examined during development of oocytes and embryos. Surprisingly, NGF mRNA was found in the oocyte; it is present in small previtellogenic as well as in fully grown oocytes. NGF mRNA, passed to the embryo at fertilization, is degraded before the gastrula stage and starts accumulating again around the stage of the neurula. The association of NGF mRNA with polysomes is indicative of NGF synthesis during oogenesis. In fact, by using antibodies against mouse NGF it was possible to reveal NGF molecules present as precursors. These molecules accumulate during oogenesis and are maintained in the embryos up to the blastula stage; a very faint band corresponding to a smaller size peptide is sometimes detected. A maternal role for the NGF can be proposed, although a possible activity of NGF in the oocyte cannot be ruled out.     

Three-dimensional organization and dynamic changes of the actin cytoskeleton in embryo sacs ofZea mays andTorenia fournieri

Summary Actin organization was observed inm-maleimidobenzoic acid N-hydroxysuccinimide ester(MBS)-treated maize embryo sacs by confocal laser scanning microscopy. The results revealed that dynamic changes of actin occur not only in the degenerating synergid, but also in the egg during fertilization. The actin filaments distribute randomly in the chalazal part of the synergid before fertilization; they later become organized into numerous aggregates in the chalazal end after pollination. The accumulation of actin at this region is intensified after the pollen tube discharges its contents. Concurrently, actin patches have also been found in the cytoplasm of the egg cell and later they accumulate in the cortical region. To compare with MBS-treated maize embryo sacs, we have performed phalloidin microinjection to label the actin cytoskeleton in living embryo sacs ofTorenia fournieri. The results have extended the previous observations on the three-dimensional organization of the actin arrays in the cells of the female germ unit and confirm the occurrence of the actin coronas in the embryo sac during fertilization. We have found that there is an actin cap occurring near the filiform apparatus after anthesis. In addition, phalloidin microinjection into the Torenia embryo sac has proved the presence of intercellular actin between the cells of the female germ unit and thus confirms the occurrence of the actin coronas in the embryo sac during fertilization. Moreover, actin dynamic changes also take place in the egg and the central cell, accomplished with the interaction between the male and female gametes. The actin filaments initially organize into a distinct actin network in the cortex of the central cell after anthesis; they become fragmented in the micropylar end of the cell after pollination. Similar to maize, actin patches have also been observed in the egg cortex after pollination. This is the first report of actin dynamics in the living embryo sac. The results suggest that the actin cytoskeleton may play an essential role in the reception of the pollen tube, migration of the male gametes, and even gametic fusion.     

Cytoskeletal pattern changes during branch formation in a centrifugedAdiantum protonema

A protonemal branch was induced on a side wall of a fern filamentous protonema by cell centrifugation and subsequent polarized-red light irradiation as described in a previous paper (Wada 1995, J. Plant Res. 108: 501–509). Changes in microtubule (MT) and microfilament (MF) patters during the branch development were observed under fluorescence microscopy. A ring-like band of cortical MTs (MT-ring) and MFs similar to a preprophase band or a subapical ring structure (Murataet al. 1987) appeared transiently at the future branching site before cell swelling, the first visible step of branch formation. At this stage, the nucleus was located far from the branching site and the MT-ring appeared to be connected to the nucleus by endoplasmic MFs as well as with endoplasmic MTs. The MT-ring disappeared when cell wall swelling occurred. When the cell wall swelling began, a fan-like pattern of cortical MTs emanating from the new growing tip was established and the MTs reached the opposite flank of the protonema. When a new branch started to elongate and the nucleus moved into the branch, a faint subapical ring of MTs appeared at the subapical part of the new branch. Strands of MTs and MFs emanating from the nuclear front end reached a part of the subapical ring.     

La protein is associated with terminal oligopyrimidine mRNAs in actively translating polysomes

La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines. In the nucleus it has a role in the metabolism of several small RNAs. A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation. The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet. Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5' end, which is necessary for the regulation of their translation. La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo. Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes. Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40-60 S. The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes. Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells.     

Ultrastructure of the “fringe-layer”, the innermost epidermis of cotton seed coats

Summary The inner epidermis of the inner integument of cotton seed coats (fringe-layer) and the cuticles between this cell layer and the nucellus were examined in the light and electron microscope at different times of their development. The cells of the fringe-layer contain only small vacuoles and their cytoplasm is densely packed with organelles and free and membrane-bound polysomes. The lateral walls contain many plasmodesmata. At the time when the fruit capsules stop growing, the fringe-cells produce a cell wall labyrinth, resembling that of transfer cells. The cell wall labyrinth is restricted to the lateral walls. The differentiated state of the fringe-cells is short-lived. At about the time of elaboration of the cell wall labyrinth most of them become progressively vacuolated, lignify, and lose their cytoplasmic constituents. The development of the fringe-layer is well correlated with other developmental events in the inner integument, but not with the filling of embryo and endosperm with reserve substances.At anthesis, the fringe-layer and nucellus are covered by a thin cuticle proper of about 20 nm. After anthesis, the nucellar cells start to produce a cuticular layer of considerable, but variable, thickness (0.25–2.5 m), containing a polysaccharide network.In drying seeds the cells of the fringe-layer disrupt. The thin outer tangential wall remains attached to the seed coat. The rest of the cell, together with the cuticles and the collapsed cells of the nucellus, form a protective layer around embryo and endosperm, remaining attached to the seed coat at the chalazal end.