Ooplasmic segregation and axis formation in the polychaeteNereis virens embryo

Ooplasmic segregation is of great importance in the development of Annelida. The mechanisms of this process are very diverse in different groups of polychaetes, oligochaetes, and leeches (Fernandezet al., 1998). Ooplasmic segregation inNereis virens is connected with the first meiotic spindle formation and animal-vegetative axis appearance. Spherical polyaxial symmetry of the oocyte transforms into radial stratified symmetry in the course of ooplasmic segregation. There are two main steps of ooplasmic segregation inNereis virens. The first step begins after the cortical reaction when the central clear cytoplasm reaches the surface of the oocyte. The movement of the cytoplasm is sensitive to nocodazole, colchicine, and cytochalasin B and appears to be mediated by microtubules and, partly, by microfilaments. The second step is not sensitive to the microtubule inhibitors and is mediated mainly by actin filaments. Ooplasmic segregation inNereis virens may be considered as a primitive form of ooplasmic segregation in Annelida.     

Ooplasmic segregation of the myoplasmic actin network in stratified ascidian eggs

Summary Ooplasmic segregation in ascidians includes the movement of the myoplasm, a pigmented cytoplasmic region thought to be involved in the determination of the embryonic muscle and mesenchyme cell lineages, into the vegetal hemisphere of the egg. A myoplasmic cytoskeletal domain (MCD), composed of a cortical actin network (the PML) and an underlying filamentous lattice extending deep into the cytoplasm, is present in this region. The MCD gradually recedes into the vegetal hemisphere during ooplasmic segregation. It has been proposed that the segregation of the myoplasm is mediated by the contraction of the PML. To test this possibility we have examined ooplasmic segregation in eggs in which the internal parts of the MCD were separated from the PML by centrifugal force. Transmission and scanning electron microscopy of eggs extracted with Triton X-100 showed that the PML remained intact when the internal portions of the MCD were displaced and stratified by centrifugation. When stratified eggs were fertilized there were no rearrangements of the visible cytoplasmic inclusions, but the cellular deformations and the recession of the PML characteristic of ooplasmic segregation occurred as usual. The results indicate that the recession of the PML occurs independently of the internal constituents of the MCD and suggest that PML contraction is the motive force for ooplasmic segregation.     

Interactions between cytoskeletal components during myoplasm rearrangement in ascidian eggs

Ooplasmic segregation in ascidian eggs consists of two phases of cytoplasmic movement, the first phase is mediated by the microfilament system and the second is mediated by the microtubule system. Recently, two novel proteins, p58 and myoplasmin-C1, which are localized to the myoplasm, were suggested to have important roles in muscle differentiation. In order to analyze the molecular mechanisms underlying ooplasmic segregation, the interactions between actin, tubulin, p58 and myoplasmin-C1 were examined. During the first segregation, microtubule meshwork in the unfertilized egg disappeared. At the second segregation, a novel structure of the microtubules that extended from the sperm aster and localized in the cortical region of the myoplasm was found. Moreover, uniform distribution of the cortical actin filament was observed at the second segregation. During the course of myoplasm rearrangement, p58 and myoplasmin-C1 are colocalized and can form a molecular complex in vitro. This complex of p58 and myoplasmin-C1 is a good candidate for a cytoskeletal component of the myoplasm, and is likely to be involved in the correct distribution of cytoplasmic determinants.     

Ooplasmic segregation in theTubifex egg: Mode of pole plasm accumulation and possible involvement of microfilaments

Summary Ooplasmic segregation, i.e. the accumulation of pole plasm in theTubifex egg, consists of two steps: (1) Cytoplasm devoid of yolk granules and lipid droplets migrates toward the egg periphery and forms a continuous subcortical layer around the whole egg; (2) the subcortical cytoplasm moves along the surface toward the animal pole in the animal hemisphere and toward the vegetal pole in the vegetal hemisphere, and finally accumulates at both poles of the egg to form the animal and vegetal pole plasms. Whereas the subcortical layer increases in volume during the first step, it decreases during the second step. This is ascribed to the compact rearrangement in the subcortical layer of membraneous organelles such as endoplasmic reticulum and mitochondria. The number of membraneous organelles associated with the cortical layer increases during the second step. Electron microscopy reveals the presence of microfilaments not only in the cortical layer but also in the subcortical layer. Subcortical microfilaments link membraneous organelles to form networks; some are associated with bundles of cortical microfilaments. The thickness of the cortical layer differs regionally. The pattern of this difference does not change during the second step. On the other hand, the subcortical cytoplasm moves ahead of the stationary cortical layer. The accumulation of pole plasm is blocked by cytochalasin B but not by colchicine. The first step of this process is less sensitive to cytochalasin B than the second step, suggesting that these two steps are controlled by differnt mechanisms. The mechanical aspects of ooplasmic segregation in theTubifex egg are discussed in the light of the present observations.     

Morphogenetic Analysis of Spiralian Development

Pattern formation in molluscs is illustrated by the exampleof the larval head pattern of gastropods. The egg cell at thebeginning of development is provided with a spatial patternof developmental factors lying in the surface membrane, by whichthe main axes of the future embryo and the frame of referenceof ooplasmic segregation are determined. Ooplasmic segregationleads to a gradient-like distribution of pole plasm substancealong the main axis and to the formation of RNA-rich granulain the vegetative cells, which play a part in the inductionof bilateral symmetry in the animal hemisphere. The patternof larval head organs arises by interaction of the axial gradientand a dorsoventral concentration gradient of an inductive substance.     

Cytoskeletal mechanisms of ooplasmic segregation in annelid eggs

Annelid embryos are comprised of yolk-deficient animal and yolk-filled vegetal blastomeres. This "unipolar" organization along the animal-vegetal axis (in terms of ooplasmic distribution) is generated via selective segregation of yolk-free, clear cytoplasm to the animal blastomeres. The pathway that leads to the unipolar organization is different between polychaetes and clitellates (i.e., oligochaetes and hirudinidans). In polychaetes, the clear cytoplasm domain, which is established through ooplasmic segregation at the animal side of the egg, is simply cut up by unequal equatorial cleavage. In clitellates, localization of clear cytoplasm to animal blastomeres is preceded by unification of the initially separated polar domains of clear cytoplasm, which result from bipolar ooplasmic segregation. In this article, I have reviewed recent studies on cytoskeletal mechanisms for ooplasmic localization during early annelid development. Annelid eggs accomplish ooplasmic rearrangements through various combinations of three cytoskeletal mechanisms, which are mediated by actin microfilaments, microtubules and mitotic asters, respectively. One of the unique features of annelid eggs isthat a homologous process is driven by distinct cytoskeletal elements. Annelid eggs may provide an intriguing system to investigate not only mechanical aspects of ooplasmic segregation but also evolutionary divergence of cytoskeletal mechanisms that operate in a homologous process.     

A Cytological Analysis of Differentiation Without Cleavage in Cytochalasin B- and Colchicine-Treated Embryos of Chaetopterus pergamentaceus

Chaetopterus eggs undergo characteristic ooplasmic rearrangements during development. Ooplasmic rearrangement in the absence of cell division is called differentiation without cleavage. Treatment of fertilized eggs with cytochalasin B allowed the continuation of nuclear divisions in the absence of cytoplasmic division. The ooplasmic rearrangements in uncleaved cytochalasin B-treated fertilized eggs closely paralleled those of normal development. Colchicine treatment, which blocks mitosis, arrested ooplasmic movements at a stage comparable to that of normal embryos at first cleavage. Neither drug eliminated the segregation between hyaloplasm and endoplasm, even though colchicine prevented the later rearrangements. Localizing movements are therefore dependent upon normal microtubule function, but not on microfilament function. The maintenance of localized materials does not seem to depend exclusively on either of these organelles.     

Preparation, Properties, and Composition of Nereis vitellin, the Yolk Protein of the Annelid, Nereis virens

In the literature, oogenesis in nereid annelids is considered to be a model system because, unlike other system studied, nereid oocytes are thought to synthesize the bulk of their yolk protein themselves. As the first step to test the validity of this hypothesis, nereid yolk protein was characterized biochemically. Vitellin, the main fraction of the soluble yolk proteins, was prepared from Nereis virens oocytes. Preparation, purification, and some physical characteristics of this green-colored protein Nereis vitellin are described. The molecular weight was determined by gel chromatography as 420,000 daltons. With regard to the amino acid composition, Nereis vitellin was found to resemble both insect vitellins and an average protein, as defined by other authors. Methionine and cysteine were found in traces only. By staining procedures, Nereis vitellin was characterized as lipoglycoprotein. Nereis vitellin was also prepared from the coelomic fluid of gravid females of Nereis virens .     

Signaling pathway from [Ca2+]i transients to ooplasmic segregation involves small GTPase rho in the ascidian egg

Intracellular Ca2+ transients occur at fertilization in the eggs of all animal species and are thought to be critical for the initiation of several events in egg activation. The rho family of small GTPases are known to organize and maintain the actin filament-dependent cytoskeleton, and rho is involved in the control mechanism of cytokinesis. In the ascidian Ciona savignyi, the first step of ooplasmic segregation observed just after fertilization is cortical contraction with egg deformation, mediated by the cortical actin filaments. C3 exoenzyme, a rho-specific inhibitor, did not affect the pattern of [Ca2+]i transients in the ascidian egg, but inhibited ooplasmic segregation and cytokinesis at the first cleavage. Injection of inositol 1,4,5-trisphosphate or treatment of Ca2+ ionophore induced deformation of the egg and extrusion of the first polar body, but these phenomena did not occur in the C3 exoenzyme-injected egg. These results suggest that rho proteins are involved in egg deformation, ooplasmic segregation and cytokinesis downstream of the [Ca2+]i transients.     

Effects of cytoskeletal inhibitors on ooplasmic segregation and microtubule organization during fertilization and early development in the ascidian Molgula occidentalis

The effects of microtubule and microfilament inhibitors on ooplasmic segregation and microtubule organization were examined during fertilization, parthenogenetic activation, and early development in the ascidian Molgula occidentalis. At fertilization the egg cortex contracts as the first phase movement and shortly after mitochondria migrate as the myoplasmic crescent develops in the second phase. The microtubule inhibitors colcemid and nocodazole inhibit the second phase, but not the first phase, of ooplasmic segregation. The microfilament inhibitor cytochalasin E has the reciprocal effect of inhibiting the first, but not the second, phase. It appears that sperm may initially bind at any site on the egg surface and that the contractile activities at the first phase and during polar body formation occur independent of the microtubule system. Since the second phase migration occurs as the sperm astral microtubules assemble and since microtubule, but not microfilament, inhibitors arrest this aspect of ooplasmic segregation, microtubules appear necessary for mitochondrial migration. These results demonstrate that the two phases of ascidian ooplasmic segregation are mediated by different systems, the first by microfilaments and the second by microtubules. The microtubule and microfilament systems appear to operate independent of one another and their combined actions result in the completion of ooplasmic segregation. A model is proposed in which the cortical contraction following fertilization is important not only as the motive force for the first phase movement but also as a method to unite the myoplasm with the entering sperm which can initially bind anywhere on the egg surface. The association between myoplasmic components and the growing sperm aster would ensure that the migration and the spatial distribution of myoplasm in the second phase results in the formation of the myoplasmic crescent.     

Effect of Colchicine and cytochalasin B on ooplasmic segregation of ascidian eggs

Summary Ooplasmic segregation inPhallusia mammillata was completed 3 to 5 min after fertilization. Colchicine, which completely stopped nuclear divisions, did not inhibit segregation. Cytochalasin B, which prevented cleavage at a low concentration (0.2 g/ml) inhibited segregation only at a concentration at least five times higher. The action of these drugs leads to the conclusion that ooplasmic segregation does not depend on an assembly of microtubules or on microfilaments which are involved in cell division.This work was performed at the Station Zoologique Villefranche-sur mer (Director: Prof. P. Bougis). The work was supported by a grant A.T.P. of C.N.R.S.     

Dynamics of the actin microfilament system in the Tubifex egg during ooplasmic segregation

Following the second polar body formation (PBF), the Tubifex egg undergoes ooplasmic segregation consisting of two steps, i.e., centrifugal migration of membranous organelles forming a subcortical ooplasmic layer and then movements of these organelles along the egg surface. The present investigation was undertaken to examine the microfilament organization in eggs during these ooplasmic rearrangements. Microfilaments throughout the egg are identified as actin by their reversible heavy meromyosin binding. Before the second PBF, a distinct network of actin filaments is present in the endoplasmic region. It is disorganized during the second PBF; short actin filaments are caused to aggregate with membranous organelles. Following the second PBF, similar short filaments become localized in the subcortical layer but not in the underlying yolky region. However, it is not until 50-60 min after the second PBF that an elaborate actin network is established in the subcortical layer. The cortex contains a sheet-like lattice of actin filaments. It is thickest around the animal pole, and tapes toward the equator of the egg. At about 90 min after the second PBF, this polarized distribution of cortical filaments becomes more pronounced as the result of their movements. Chronologically, subcortical actin network formation and cortical reorganization correspond to the later portion of the first step and the earlier portion of the second step of ooplasmic segregation, respectively. These findings are discussed in terms of ooplasmic movements and rearrangements.     

Bipolar segregation of mitochondria,actin network,and surface in the Tubifex egg: Role of cortical polarity

The role of the cortex in ooplasmic segregation of the yolky eggs of Tubifex has been studied by epifluorescence microscopy. Living eggs labeled with rhodamine 123 and fine carbon particles placed on the surface showed that, following the second polar body formation, the egg surface cosegregates with subcortical mitochondria in a bipolar fashion, viz. toward the animal and vegetal poles in the animal and vegetal hemispheres, respectively. The egg surface of each pole moves spirally while the equatorial surface appears to remain stationary during this process. The rhodamine-phalloidin staining of whole eggs reveals that actin networks cosegregate with mitochondria. Isolated cortices which were stained with rhodamine-phalloidin demonstrated that cortical actin is organized bipolarly and that, during ooplasmic segregation, it undergoes reorganization directed toward both poles of the egg. The cortical polarity expressed as actin organization is not disrupted by centrifugal force sufficient to stratify the egg cytoplasm into five layers. The surface of a centrifuged egg moves according to the original cortical polarity. This surface movement is accompanied by the reorganization of cortical actin which appears to be identical to that in intact eggs. Other centrifugation experiments have demonstrated that the connection of the subcortical cytoplasm to the cortex is resistant to a centrifugal force of up to 650g. The nature of cortical polarity and its role in ooplasmic segregation are discussed in the light of the present results.     

The cortical contraction related to the ooplasmic segregation inCiona intestinalis eggs

Summary The egg cytoplasm of ascidian,Ciona intestinalis, segregates towards both the animal and vegetal poles within a few minutes of fertilization or parthenogetic activation with ionophore A23187. A constriction appears first on the egg surface near the animal pole and then moves to the vegetal pole. Carmine granules and spermatozoa attached to the egg surface move towards the vegetal pole with the movement of the constriction. Microvilli, which are distributed uniformly in unfertilized egg, disappear on the animal side of the constriction and became more dense on the vegetal side of the constriction. Transmission electron microscopy revealed that sub-cortical cytoplasm, containing numerous mitochondria and sub-cortical granules, moves towards the vegetal pole with the movement of the constriction and then concentrates into a cytoplasmic cap at the vegetal pole. An electron-dense layer appears in the cortex of the cap. The ooplasmic segregation and the cortical contraction were inhibited by cytochalasin B and induced by ionophore A23187. These observations suggest that ooplasmic segregation is caused by the cortical contraction which is characterised by a surface constriction and by the formation of an electron-dense layer.     

Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos

A cDNA library prepared from fertilized eggs of the ascidian Halocynthia roretzi was screened for prelocalized mRNAs in the early embryo by means of whole-mount in situ hybridization using a digoxigenin-labeled antisense RNA of each clone. Random mass screening of 150 cDNAs in a fertilized egg yielded six different clones which showed mRNA localization in the posterior-vegetal cytoplasm of the 8-cell embryo. An in situ hybridization study of the detailed spatial distribution of each mRNA in embryos of various stages revealed that there are, in contrast to the identical localization in embryos after the 16-cell stage, two distinct patterns of RNA distribution at earlier stages. One is colocalization with the myoplasm from the prefertilization stage to the 8-cell stage (type I postplasmic RNAs). The other is delayed accumulation of RNA at the posterior-vegetal cytoplasm after fertilization (type II postplasmic RNAs). We found that both types of RNAs associate with the cytoskeleton, but that they show different sensitivities to inhibitors of the cytoskeleton; translocation of the type I RNAs is dependent upon microfilaments during the first phase of ooplasmic segregation and dependent upon microtubules during the second phase of segregation, whereas translocation of the type II RNAs is dependent upon microfilaments throughout ooplasmic segregation. These results show that there are two pathways for the localization of the RNAs at the posterior-vegetal cytoplasm in the 8-cell embryo of the ascidian H. roretzi.     

Intercellular cytoplasm transport during oogenesis of the moth midge, Tinearia alternata Say (Diptera: Psychodidae)

Polytrophic ovaries of the nematocerous dipteran, Tinearia alternata Say consists of several developmentally synchronized ovarioles each housing only one functional egg chamber with 15 nurse cells and an oocyte. At the early stages of previtellogenesis the nurse cells become polyploid and synthetically active. Their nuclei contain polytene chromosomes and prominent nucleoli. With the advance of previtellogenic growth the nurse cell cytoplasm is loaded with the growing number of ribosomes and contain perinuclear nuage material, mitochondria, electron dense bodies and aggregations of endoplasmic reticulum. All these organelles are transported into the oocyte thanks to the massive and rapid flow of the nurse cell cytoplasmic contents. Nurse cell-oocyte transport is mediated by actin cytoskeleton. Prior to the rapid cytoplasm transfer, F-actin network is associated with the nurse cell membranes while tiny bundles of microfilaments form actin baskets connected with ring canals. Nurse cells in Tinearia lack an extensive scaffold of radially oriented, F-actin bundles (cables) that would tether their nuclei in place, thus preventing ring canals from plugging. The way the nuclei are anchored to their central positions within the cells remains unclear. Towards the final stages of oogenesis nurse cells are almost devoid of cytoplasm and degenerate. Although their nuclei undergo dramatic morphological transformations, typical hallmarks of apoptotic pathway could not be clearly observed. Rapid ooplasmic streaming does not occur.     

Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg

During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiIC16(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER- poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. Dev. Biol. 153:259-271). This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.     

Confocal and video imaging of cytoskeleton dynamics in the leech zygote

Ooplasmic segregation in the late interphase zygote of the leech Theromyzon trizonare is accomplished by reorganization of an ectoplasmic cytoskeleton formed by polar rings and meridional bands. The dynamic properties of this cytoskeleton were explored by time-lapse confocal and video microscopy. Cytoskeleton assembly was investigated in zygotes pulse-labeled with microinjected fluorophore-tagged or biotin-tagged dimeric tubulin and G-actin. Cytoskeleton disassembly was studied by comparing the linear dimensions of the cytoskeleton at different time points during late interphase. The relative distributions of F- and-G-actin were determined after microinjection of rhodamine-labeled actin and fluorescein-labeled DNase I. Results showed that labeled precursors were readily incorporated into a network of microtubules or actin filaments. Bipolar translocation of the rings and meridional bands was accompanied by the rapid assembly and disassembly of microtubules and actin filaments. Because labeled microtubules and microfilaments gradually decreased, the rate of cytoskeleton disassembly was greater than the rate of cytoskeleton assembly. Hence, ooplasmic segregation was accompanied by the rapid turnover of cytoskeletal components. Co-distribution of F- and-G-actin during mid and late interphase may favor polymer-monomer interchange. We conclude that cytoskeleton reorganization during foundation of cytoplasmic domains can be conveniently studied in the live leech zygote after microinjection of labeled precursors.     

卵胞质移植的研究进展

许多研究表明,线粒体对卵母细胞的受精和胚胎发育有显著影响,卵胞质中线粒体DNA含量和ATP含量的减少,以及线粒体DNA缺失均能降低卵母细胞的受精和胚胎发育,是老龄妇女和老龄动物生育率下降的重要原因之一。卵胞质移植技术能有效改善老龄卵母细胞的受精能力和早期胚胎的发育能力,在人类已有健康后代出生,它已成为人类辅助生殖生物技术和动物克隆研究的新热点。但是,卵胞质转移也可能会导致线粒体DNA异质,即供体和受体的线粒体DNA同时存在于后代体内。目前,人们对于转入的异质卵胞质中对胚胎的发生和发育造成影响的因素并不完全了解。通过卵胞质移植研究概况、卵胞质与受精和胚胎发育、异种线粒体DNA遗传方式和卵胞质转移遗传物质的检测4个方面对卵胞质移植技术进行讨论。     

Regulation of ooplasmic sodium and potassium activities in developing locust eggs

Ooplasmic activities of potassium and sodium were measured with ion sensitive microelectrodes before and during the period of maximal water uptake which occurs 3–5 days after oviposition for eggs incubated at 37°C. Potassium activity increased from 84 mM in eggs before fertilization at 118 mM in eggs 1 day after fertilization (d1). Sodium activity increased from 8 mM to 29 mM over the same period. These changes exceeded those predicted from the decrease in water content (8%) during the first day after oviposition. Between d1 and d3, potassium and sodium activities decreased to values predicted on the basis of the 88% increase in egg water content. Although water content increased an additional 46% between d3 and d5, ooplasmic sodium activity remained constant at 11 mM and potassium activity increased from 64 mM to 74 mM during this time. Declines in concentrations of sodium and potassium measured in whole eggs by atomic absorption spectrometry mirrored the increase in egg water content. The results suggest that regulation of ooplasmic sodium and potassium activities is accomplished by release of these ions from internal stores, possibly the york spheres. © 1992 Wiley-Liss, Inc.