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1.
Cytokeratin intermediate filaments are prominent constituents of developing Xenopus oocytes and eggs, forming radial and cortical networks. In order to investigate the dynamics of the cortical cytokeratin network, we expressed EGFP-tagged Xenopus cytokeratin 1(8) in oocytes and eggs. The EGFP-cytokeratin co-assembled with endogenous partner cytokeratin proteins to form fluorescent filaments. Using time-lapse confocal microscopy, cytokeratin filament assembly was monitored in live Xenopus oocytes at different stages of oogenesis, and in the artificially-activated mature egg during the first cell cycle. In stage III to V oocytes, cytokeratin proteins formed a loose cortical geodesic network, which became more tightly bundled in stage VI oocytes. Maturation of oocytes into metaphase II-arrested eggs induced disassembly of the EGFP-cytokeratin network. Imaging live eggs after artificial activation allowed us to observe the reassembly of cytokeratin filaments in the vegetal cortex. The earliest observable structures were loose foci, which then extended into curly filament bundles. The position and orientation of these bundles altered with time, suggesting that forces were acting upon them. During cortical rotation, the cytokeratin network realigned into a parallel array that translocated in a directed manner at 5 microm/minute, relative to stationary cortex. The cytokeratin filaments are, therefore, moving in association with the bulk cytoplasm of the egg, suggesting that they may provide a structural role at the moving interface between cortex and cytoplasm.  相似文献   

2.
In Xenopus, one of the most dramatic events during oocyte maturation is the breakdown of the oocyte's asymmetrically organized system of cytokeratin-type intermediate filaments. Following oocyte maturation in vitro, we found that (1) the breakdown of cytokeratin filament organization proceeds in an animal to vegetal direction, (2) cytokeratin filament breakdown occurs normally in enucleated oocytes and so is independent of nuclear components, and (3) the injection of maturation-promoting factor (MPF) induces the breakdown of cytokeratin filaments. While the MPF-induced breakdown of the nuclear envelope is independent of new protein synthesis, the MPF-induced breakdown of cytokeratin filament organization requires the translation of maternal mRNAs. These results strongly suggest that the factors regulating cytokeratin reorganization in the oocyte are distinct from those involved in the breakdown of the nuclear envelope.  相似文献   

3.
To study vimentin filament organization in vivo we injected Xenopus oocytes, which have no significant vimentin system of their own, with in vitro-synthesized RNAs encoding Xenopus vimentins. Exogenous vimentins were localized primarily to the cytoplasmic surface of the nucleus and to the subplasma membrane "cortex." In the cortex of the animal hemisphere, wild-type vimentin forms punctate structures and short filaments. In contrast, long anastomosing vimentin filaments are formed in the vegetal hemisphere cortex. This asymmetry in the organization of exogenous vimentin is similar to that of the endogenous keratin system (Klymkowsky, M. W., L. A. Maynell, and A. G. Polson. 1987. Development (Camb.). 100:543-557), which suggests that the same cellular factors are responsible for both. Before germinal vesicle breakdown, in the initial stage of oocyte maturation, large vimentin and keratin filament bundles appear in the animal hemisphere. As maturation proceeds, keratin filaments fragment into soluble oligomers (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787-797), while vimentin filaments remain intact and vimentin is hyperphosphorylated. To examine the role of MPF kinase in the M-phase reorganization of vimentin we deleted the conserved proline of vimentin's single MPF-kinase site; this mutation had no apparent effect on the prophase or M-phase behavior of vimentin. In contrast, deletion of amino acids 19-68 or 18-61 of the NH2-terminal "head" domain produced proteins that formed extended filaments in the animal hemisphere of the prophase oocyte. We suggest that the animal hemisphere cortex of the prophase oocyte contains a factor that actively suppresses the formation of extended vimentin filaments through a direct interaction with vimentin's head domain. During maturation this "suppressor of extended filaments" appears to be inactivated, leading to the formation of an extended vimentin filament system.  相似文献   

4.
5.
The asymmetric distribution of cellular components is an important clue for understanding cell fate decision during embryonic patterning and cell functioning after differentiation. In C. elegans embryos, PAR-3 and aPKC form a complex that colocalizes to the anterior periphery of the one-cell embryo, and are indispensable for anterior-posterior polarity that is formed prior to asymmetric cell division. In mammals, ASIP (PAR-3 homologue) and aPKCgamma form a complex and colocalize to the epithelial tight junctions, which play critical roles in epithelial cell polarity. Although the mechanism by which PAR-3/ASIP and aPKC regulate cell polarization remains to be clarified, evolutionary conservation of the PAR-3/ASIP-aPKC complex suggests their general role in cell polarity organization. Here, we show the presence of the protein complex in Xenopus laevis. In epithelial cells, XASIP and XaPKC colocalize to the cell-cell contact region. To our surprise, they also colocalize to the animal hemisphere of mature oocytes, whereas they localize uniformly in immature oocytes. Moreover, hormonal stimulation of immature oocytes results in a change in the distribution of XaPKC 2-3 hours after the completion of germinal vesicle breakdown, which requires the kinase activity of aPKC. These results suggest that meiotic maturation induces the animal-vegetal asymmetry of aPKC.  相似文献   

6.
Mature ascidian oocytes are arrested in metaphase of meiosis I (Met I) and display a pronounced animal-vegetal polarity: a small meiotic spindle lies beneath the animal pole, and two adjacent cortical and subcortical domains respectively rich in cortical endoplasmic reticulum and postplasmic/PEM RNAs (cER/mRNA domain) and mitochondria (myoplasm domain) line the equatorial and vegetal regions. Symmetry-breaking events triggered by the fertilizing sperm remodel this primary animal-vegetal (a-v) axis to establish the embryonic (D-V, A-P) axes. To understand how this radial a-v polarity of eggs is established, we have analyzed the distribution of mitochondria, mRNAs, microtubules and chromosomes in pre-vitellogenic, vitellogenic and post-vitellogenic Germinal Vesicle (GV) stage oocytes and in spontaneously maturing oocytes of the ascidian Ciona intestinalis. We show that myoplasm and postplasmic/PEM RNAs move into the oocyte periphery at the end of oogenesis and that polarization along the a-v axis occurs after maturation in several steps which take 3-4 h to be completed. First, the Germinal Vesicle breaks down, and a meiotic spindle forms in the center of the oocyte. Second, the meiotic spindle moves in an apparently random direction towards the cortex. Third, when the microtubular spindle and chromosomes arrive and rotate in the cortex (defining the animal pole), the subcortical myoplasm domain and cortical postplasmic/PEM RNAs are excluded from the animal pole region, thus concentrating in the vegetal hemisphere. The actin cytoskeleton is required for migration of the spindle and subsequent polarization, whereas these events occur normally in the absence of microtubules. Our observations set the stage for understanding the mechanisms governing primary axis establishment and meiotic maturation in ascidians.  相似文献   

7.
In zebrafish, as in many animals, maternal dorsal determinants are vegetally localized in the egg and are transported after fertilization in a microtubule-dependent manner. However, the organization of early microtubules, their dynamics and their contribution to axis formation are not fully understood. Using live imaging, we identified two populations of microtubules, perpendicular bundles and parallel arrays, which are directionally oriented and detected exclusively at the vegetal cortex before the first cell division. Perpendicular bundles emanate from the vegetal cortex, extend towards the blastoderm, and orient along the animal-vegetal axis. Parallel arrays become asymmetric on the vegetal cortex, and orient towards dorsal. We show that the orientation of microtubules at 20 minutes post-fertilization can predict where the embryonic dorsal structures in zebrafish will form. Furthermore, we find that parallel microtubule arrays colocalize with wnt8a RNA, the candidate maternal dorsal factor. Vegetal cytoplasmic granules are displaced with parallel arrays by ~20°, providing in vivo evidence of a cortical rotation-like process in zebrafish. Cortical displacement requires parallel microtubule arrays, and probably contributes to asymmetric transport of maternal determinants. Formation of parallel arrays depends on Ca(2+) signaling. Thus, microtubule polarity and organization predicts the zebrafish embryonic axis. In addition, our results suggest that cortical rotation-like processes might be more common in early development than previously thought.  相似文献   

8.
During meiotic maturation, the cortical cytokeratin filament system of the Xenopus oocyte disappears (Klymkowsky, M. W., and L. A. Maynell. 1989. Dev. Biol. 134:479). Here we demonstrate that this disappearance results from the severing of cytokeratin filaments into a heterogenous population of oligomers, with S- values ranging from 12S and greater. Cytokeratin filament severing correlates with the hyperphosphorylation of the type II cytokeratin of the oocyte. Both the severing of cytokeratin filaments and cytokeratin hyperphosphorylation are reversed by treatment with cycloheximide. These data suggest that fragmentation of cytokeratin filaments is controlled, at least in part, by the phosphorylation of the type II cytokeratin, and that the cytokeratin kinase activity responsible is biosynthetically labile. Cytokeratin filaments have been suggested to anchor the maternal mRNA Vg1 to the vegetal cortex of the oocyte (Pondel, M., and M. L. King. 1988. Proc. Natl. Acad. Sci. USA. 85:7216). By injecting fractions containing active maturation promoting factor or a purified, mutant cyclin protein, we find that the bulk of the Vg1 mRNA in the oocyte can be solubilized under conditions that block the fragmentation of cytokeratin filaments, and that the fragmentation of cytokeratin filaments itself leads to the solubilization of only a minor fraction of the Vg1 mRNA. Thus, at best, cytokeratin filaments directly anchor only a minor fraction of the Vg1 mRNA in the oocyte. Moreover, factors distinct from maturation promoting factor appear to be required for the complete solubilization of Vg1 mRNA during oocyte maturation.  相似文献   

9.
The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos.  相似文献   

10.
A frozen section technique for frog oocytes was developed without using any organic solvent. It was applied to examine the distribution of acidic glycosphingolipids (ganglioside GM1 and sulfatide) in Xenopus oocytes, eggs and embryos by indirect immunofluorescence microscopy with specific monoclonal antibodies against the acidic glycolipids. Although glycolipids are generally present on the cell surface, GM1 and sulfatide were distributed in the cytoplasm of animal and vegetal hemispheres, respectively, of the fully grown oocytes and oviposited and fertilized eggs. In blastula, GM1 was present on the cell boundaries and in the Golgi of the blastomeres of animal hemisphere and marginal zone, whereas the staining of the outermost layer of animal blastomeres became faint or negligible at stage 9. Sulfatide in blastula was still observed in vegetal blastomeres. In gastrula, GM1 was distributed in the inner layer of ectoderm and the involuting mesoderm. In neurula, GM1 was concentrated in the dorsal midline including the closing neural tube, notochord and somites, while sulfatide was present in endoderm. The unique distribution of GM1 and sulfatide in oocytes, eggs and early embryos may help to elucidate one aspect of the biochemical bases laid on the animal–vegetal polarity.  相似文献   

11.
Immature oocytes or mature eggs of starfish were centrifuged in a sucrose density gradient. They were then separated into two fractions of fragments, nucleate light fragments and anucleate heavy fragments. Vital-staining experiments showed that the oocytes were elongated along the animal-vegetal (AV) axis during the centrifugation in a contrast to centrifuged eggs whose centrifugal axis was not related to the AV axis. The light and heavy oocyte fragments were comprised of animal and vegetal halves of oocytes, respectively. When matured and fertilized, most of the light oocyte fragment-derived embryos failed gastrulation and developed into Dauerblastulae. Two-dimensional gel electrophoretic analysis of fragments revealed that three basic proteins were predominantly enriched in the heavy oocyte fragments but scarcely detected in the light oocyte fragments. One of these proteins, App20, was identified as a homologue of cyclophilin (peptidyl-prolyl cis-trans isomerase). The present study provides a simple means of separating a population of starfish oocytes into animal and vegetal halves, thereby enabling us to analyze any difference of components between animal and vegetal cytoplasm of the oocytes.  相似文献   

12.
The animal-vegetal axis of the oocyte of Xenopus laevis is recognizable not only by the pattern of surface pigmentation, but also by the distribution of yolk platelets, with the largest platelets (congruent to 14 microns in diameter) and 70% of the total yolk protein localized in the vegetal hemisphere. We have used fluorescent and radioactive vitellogenins (yolk protein precursors) to study the spatial and temporal patterns of yolk deposition along this axis. We find that the rate of uptake of vitellogenin is nearly uniform over the surface of vitellogenic oocytes of all sizes. Once formed, yolk platelets in the animal hemisphere move inward, around the germinal vesicle, and into the central region of the vegetal hemisphere. Yolk platelets of the vegetal hemisphere do not actively move but are slowly displaced from the surface by successive layers of younger platelets arising and enlarging near the surface. The oldest yolk platelets, which arise circumcortically at the beginning of vitellogenesis in stage II and III oocytes, eventually come to reside in the vegetal hemisphere of stage VI oocytes, in the upper portion of the cup-shaped region of largest platelets. The vegetal hemisphere thus gains the majority of yolk protein by directed intracellular transport from the animal hemisphere adding to the amount directly sequestered by the vegetal hemisphere.  相似文献   

13.
Maternally accumulated materials in Xenopus oocytes, in particular mRNAs and proteins, are considered to participate in the determination of the developmental specification of embryonic cells. In this study, a large number of monoclonal antibodies was raised against bulk oocyte antigens to examine patterns of intracellular distribution of oocyte proteins. Immunohistochemical experiments with mature oocytes showed that there are five different patterns of distribution of oocyte proteins, with enrichment on the animal side (type A1, and A2 ptoteins), vegetal side (type V1 and V2 proteins), and in the peripheral cytoplasm (type P proteins). Clear localization of type A and V antigen proteins occurred at Dumont's stages IV-VI. However, at the preceding stages, the distributions of these antigen proteins appeared to be homogeneous. By contrast, the pattern of distribution of type P protenis did not change markedly throughout oogenesis. The presence of type A and type V antigen proteins reflected the animal-vegetal axis in the cytoplasm of the mature oocyte. Furthermore, there were two boundaries of the distributions of proteins at the equatorial region, excluding or including the cytoplasm around the germinal vesicle. Thus, the cytoplasm of mature oocytes was multilayered with respect to the different proteins distributed along the animal-vegetal axis.  相似文献   

14.
Extensive arrays of microfilaments, microtubules and cytokeratin-type intermediate filaments were detected in the cortex of Strongylocentrotus droebachiensis oocytes using fluorescently labeled antibodies on both cortex and whole mount preparations. All three filament systems undergo dramatic structural reorganization during meiotic maturation of the egg. Microfilaments form a dense meshwork within the cortex of the oocyte. After meiosis, the filaments rearrange and shorten, resulting in a more loosely organized network. Both cortical microtubules and microtubules associated with a microtubule-organizing center are observed within the oocyte. After meiosis, the number and length of the cortical microtubules gradually diminish. A microtubule organizing center is found situated between the germinal vesicle and the plasma membrane in many oocytes. A network of filaments extends from the microtubule organizing center and radiates peripherally toward the germinal vesicle, presumably marking the animal pole. Cytokeratin-like intermediate filaments form a reticular network within the oocyte cortex, then solubilize during meiosis. In whole mounts of oocytes there is a single focal center of cytokeratin staining from which filaments radiate. Indirect immunofluorescence experiments, using anti-tubulin and anti-cytokeratin antibodies simultaneously, reveal the intermediate filament focal center to be localized within the microtubule organizing center. These results demonstrate the presence of a complex cortical cytoskeleton in premeiotic eggs of the sea urchin, Strongylocentrotus droebachiensis.  相似文献   

15.
Light and electron microscope methods were used to study cytokeratin expression in the recently ovulated oocytes, fertilized eggs and early embryos from the golden hamster. Two cytokeratin polypeptides (Mr 51,000 and 58,000) were detected in oocyte lysates by immunoblotting using a polyclonal antiserum to prekeratin. In the oocyte, cytokeratin occurred as patchy aggregates consisting of short anastomosing 10-nm filaments that formed tight meshworks distributed throughout the cytoplasm. After fertilization the aggregates appeared to merge, becoming larger and concentrated at the cortical region. Prominent immunofluorescent fibrils were seen interconnecting the aggregates. In the 2-, 4- and 8-cell embryos, networks of cytokeratin filaments extended throughout the cortical and perinuclear regions, while aggregates progressively disappeared.  相似文献   

16.
17.
beta-Catenin has a central role in the early axial patterning of metazoan embryos. In the sea urchin, beta-catenin accumulates in the nuclei of vegetal blastomeres and controls endomesoderm specification. Here, we use in-vivo measurements of the half-life of fluorescently tagged beta-catenin in specific blastomeres to demonstrate a gradient in beta-catenin stability along the animal-vegetal axis during early cleavage. This gradient is dependent on GSK3beta-mediated phosphorylation of beta-catenin. Calculations show that the difference in beta-catenin half-life at the animal and vegetal poles of the early embryo is sufficient to produce a difference of more than 100-fold in levels of the protein in less than 2 hours. We show that dishevelled (Dsh), a key signaling protein, is required for the stabilization of beta-catenin in vegetal cells and provide evidence that Dsh undergoes a local activation in the vegetal region of the embryo. Finally, we report that GFP-tagged Dsh is targeted specifically to the vegetal cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned predominantly into vegetal blastomeres. An extensive mutational analysis of Dsh identifies several regions of the protein that are required for vegetal cortical targeting, including a phospholipid-binding motif near the N-terminus.  相似文献   

18.
The intermediate filament systems of the established epithelial cell lines HeLa and PtK2 have been characterized by electron microscopy using indirect immunoferritin labelling. The results provide a direct ultrastructural confirmation of the proposal based on indirect immunofluorescence microscopy, that vimentin and cytokeratin fibrils constitute two distinct 10 nm filament systems in much of the cell body. In agreement both with classical histology and with the finding that cytokeratins are typically present in many epithelial tissues, demosome-attached 10 nm filaments (tonofilaments) were found to be of the cytokeratin type. Vimentin, but not cytokeratin filaments were translocated into juxtanuclear caps after exposure of the cells to colcemid. Regions of the cytoplasm where the two distinct systems form mixed bundles were identified and both side-by-side arrangements and the occurrence of vimentin fibers in a sheath-like structure around a cytokeratin filament core are described. Our results emphasize that the two systems interact but differ in their organization and control.  相似文献   

19.
Polarity of the ascidian egg cortex before fertilization.   总被引:5,自引:0,他引:5  
The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.  相似文献   

20.
An animal-vegetal net ionic current identified previously using voltage probe techniques in maturing Xenopus laevis oocytes has now been investigated using noninvasive ion-selective microelectrodes. Three-dimensional fluxes of hydrogen (H(+)), potassium (K(+)), and bicarbonate (HCO(3)(-)) were characterized with respect to the developmental stage and hemisphere of the oocyte and presence of surrounding follicular tissue. Variable effluxes of H(+) and HCO(3)(-) were recorded from both the animal and vegetal hemispheres. Variable influxes and effluxes of K(+) were also observed. The equatorial region, silent by voltage probe, exhibited fluxes of H(+) and K(+). Simultaneous measurement of pairs of ions allowed correlation analysis of two ion types. Notably for H(+) and K(+) data, positive and negative correlation at animal and vegetal poles respectively offer an explanation of the unpredictable results obtained when individual ions were observed independently.  相似文献   

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