Provisional bilateral symmetry in Xenopus eggs is established during maturation

Dorsal-ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30 degree rotation of the subcortical yolk mass relative to the egg cortex. This 'rotation of symmetrisation' is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15 degrees from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90 degrees from upright during maturation in vitro, the maturation spot developed 15 degrees or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.     

Localized maternal proteins in Xenopus revealed by subtractive immunization

It has long been appreciated that the localization of cytoplasmic determinants in the egg can provide the foundation for patterning in the embryo. Differences in cell fate among the early blastomeres are thus a consequence of asymmetric distributions of informational molecules prior to fertilization. The frog egg has a single axis of asymmetry present prior to fertilization, the animal/vegetal axis, and the localization of developmental information appears to be polarized along this axis. Such developmental information can be localized as either RNA or protein; localized RNAs are well documented in the Xenopus oocyte, and some are thought to play roles in axial patterning. While it is apparent that not all of the localized maternal components are RNAs, much less is known about maternal proteins that might be localized in the egg. In the present study, we have taken a novel approach to identify localized maternal proteins within the Xenopus egg. Using a subtractive immunization strategy, we have generated monoclonal antibodies which recognize antigens that are restricted to the vegetal cortex of fertilized eggs. Analysis of biogenesis during oogenesis reveals two distinct patterns of localization to the cortex. At least three of these localized antigens are proteins, and these localized proteins could represent maternal determinants with roles in patterning.     

BMP regulates vegetal pole induction centres in early xenopus development

BACKGROUND: Bone morphogenetic protein (BMP) plays an important role in mesoderm patterning in Xenopus. The ectopic expression of BMP-4 protein hyperventralizes embryos, whereas embryos expressing a BMP-2/4 dominant-negative receptor (DNR) are hyperdorsalized. Mesoderm is initially induced in the marginal zone by cells in the underlying vegetal pole. While much is known about BMP's expression and role in patterning the marginal zone, little is known about its early role in regulating vegetal mesoderm induction centre formation. RESULTS: The role of BMP in regulating formation of vegetal mesoderm inducing centres during early Xenopus development was examined. Ectopic BMP-4 expression in vegetal pole cells inhibited dorsal mesoderm induction but increased ventral mesoderm induction when recombined with animal cap ectoderm in Nieuwkoop explants. 32-cell embryos injected with BMP-4 RNA in the most vegetal blastomere tier were not hyperdorsalized by LiCl treatment. The ectopic expression of Smad or Mix.1 proteins in the vegetal pole also inhibited dorsal mesoderm induction in explants and embryos. Expression of the BMP 2/4 DNR in the vegetal pole increased dorsal mesoderm induction and inhibited ventral mesoderm induction in explants and embryos. CONCLUSIONS: These results support a role for BMP signalling in regulating ventral vegetal and dorsal vegetal mesoderm induction centre formation during early Xenopus development.     

The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle

Embryos with no dorsal axis were obtained when more than 15% of the egg surface was deleted from the vegetal pole of the early 1-cell embryo of Xenopus laevis. The timing of the deletion in the first cell cycle was critical: dorsal-deficient embryos were obtained when the deletion began before time 0.5 (50% of the first cell cycle) whereas normal dorsal axis usually formed when the deletion was done later than time 0.8. The axis deficiency could be restored by lithium treatment and the injection of vegetal but not animal cytoplasm. Bisection of the embryo at the 2-cell stage, which is known to restore the dorsal structures in the UV-ventralized embryos, had no effect on the vegetal-deleted embryos. These results show clearly that, in Xenopus, (1) the dorsal determinants (DDs) localized in the vegetal pole region at the onset of development are necessary for dorsal axis development and (2) the DDs move from the vegetal pole to a subequatorial region where they are incorporated into gastrulating cells to form the future organizing center. A model for the early axis formation process in Xenopus is proposed.     

Fertilization stimulates an increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs

Previous experiments from our lab have suggested that the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is required for sperm-induced egg activation in Xenopus laevis. Here we measure the endogenous production of both Ins(1,4,5)P3 and PIP2 during the sperm-induced and ionomycin-induced calcium wave in the egg and find that both increase following fertilization. Ins(1,4,5)P3 increases 3.2-fold from an unfertilized egg level of 0.13 pmole per egg (0.29 microM) to a peak of 0.42 pmole per egg (0.93 microM) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P3 during the time that the Ca2+ wave is propagating across the egg suggests the involvement of Ins(1,4,5)P3 in wave propagation. This increase in Ins(1,4,5)P3 is smaller in ionomycin-activated eggs than in sperm-activated eggs, suggesting that the sperm-induced production of Ins(1,4,5)P3 involves a PIP2 hydrolysis pathway that is not simply raising intracellular Ca2+. While one might expect PIP2 levels to fall as a result of hydrolysis, we find that PIP2 actually increases 2-fold. The total lipid fraction in unfertilized egg exhibits 0.8 pmole PIP2 per egg and this increases to 1.5 pmole as the calcium wave reaches the antipode. The PIP2 concentration peaks 2 min after the completion of the calcium wave at 1.8 pmole per egg. The amount of PIP2 in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eggs in half. The vegetal hemisphere contained twice the amount of PIP2 as the animal hemisphere but it also contained twice the amount of lipid. Thus, there was an equivalent amount of PIP2 normalized to lipid in each hemisphere. Isolated animal and vegetal hemisphere cortices exhibit similar PIP2 concentrations, suggesting that the 2-fold higher total PIP2 in the vegetal half is not due to a gradient of PIP2 in the plasma membrane, but rather implies that cytoplasmic organelle membranes also contain PIP2.     

The fertilization potential provides a fast block to polyspermy in lamprey eggs

At fertilization, the membrane potential of the egg of the lamprey, Lampetra japonica, shifted rapidly from its resting value of -12 to +36 mV and gradually returned to about the same resting level (fertilization potential). The amplitude of depolarization was influenced by the external Cl- concentration and by an anion channel blocker, DIDS, indicating that the positive shift of membrane potential resulted from Cl- efflux. A similar change in membrane potential (activation potential) was observed when the unfertilized egg was pricked with a fine needle or treated with A23187 to induce parthenogenetic activation. Pricking at the animal pole region (predetermined site for sperm entry) resulted in the occurrence of an immediate activation potential and the initiation of cortical granule exocytosis. A time lag between the pricking and the occurrence of the activation potential was observed when the egg was pricked at a distance from the animal pole. In this instance, the activation potential was produced immediately before the propagating cortical granule exocytosis initiated at the pricked site reached the animal pole region. Sperm-egg fusion was blocked in eggs voltage-clamped at +20 to +40 mV and inseminated, whereas it took place in eggs clamped at -60 to 0 mV. However, most eggs clamped at +20 to +40 mV did activate, indicating that the voltage dependence of egg activation differs from that of sperm-egg fusion. Although eggs voltage-clamped at negative membrane potentials permitted multiple sperm to fuse with the egg plasma membrane, the nucleus of the fused sperm did not necessarily enter the ooplasm. We conclude that: (1) A fast electrical block against polyspermy operates in this species and is effective for about 160 sec of the onset of the positive shift; (2) the opening of Cl- channels is responsible for the potential change; (3) the channels are largely localized in the animal pole region; (4) during voltage clamp at positive potentials, eggs can be activated without sperm-egg fusion; and (5) during voltage clamp at negative potentials, sperm-egg fusion occurs, but sperm entry into the egg cytoplasm does not always proceed.     

Calcium oscillations, sperm factors and egg activation at fertilisation

When an egg is fertilised by sperm, the first intracellular signalling event observed is a large transient increase in cytoplasmic free Ca2+ ions. Elevated Ca2+ is known to play a vital role as an intracellular messenger in all cells and the Ca2+ signal occurring in the egg at fertilisation triggers the subsequent events that mediate early embryo development. In mammalian eggs, the Ca2+ response is first observed as a Ca2+ wave that initiates near the point of sperm-egg fusion, spreads across the entire egg, and then continues as a series of intracellular Ca2+ oscillations. The way in which the fertilising sperm generates the Ca2+ response in the egg has been the subject of much debate over recent years. One proposal for which there is growing evidence suggests the mechanism of egg activation at fertilisation involves the introduction of a soluble sperm protein into the egg shortly after sperm-egg fusion.     

GSK3beta/shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo

In the sea urchin embryo, the animal-vegetal axis is defined before fertilization and different embryonic territories are established along this axis by mechanisms which are largely unknown. Significantly, the boundaries of these territories can be shifted by treatment with various reagents including zinc and lithium. We have isolated and characterized a sea urchin homolog of GSK3beta/shaggy, a lithium-sensitive kinase which is a component of the Wnt pathway and known to be involved in axial patterning in other embryos including Xenopus. The effects of overexpressing the normal and mutant forms of GSK3beta derived either from sea urchin or Xenopus were analyzed by observation of the morphology of 48 hour embryos (pluteus stage) and by monitoring spatial expression of the hatching enzyme (HE) gene, a very early gene whose expression is restricted to an animal domain with a sharp border roughly coinciding with the future ectoderm / endoderm boundary. Inactive forms of GSK3beta predicted to have a dominant-negative activity, vegetalized the embryo and decreased the size of the HE expression domain, apparently by shifting the boundary towards the animal pole. These effects are similar to, but even stronger than, those of lithium. Conversely, overexpression of wild-type GSK3beta animalized the embryo and caused the HE domain to enlarge towards the vegetal pole. Unlike zinc treatment, GSK3beta overexpression thus appeared to provoke a true animalization, through extension of the presumptive ectoderm territory. These results indicate that in sea urchin embryos the level of GSKbeta activity controls the position of the boundary between the presumptive ectoderm and endoderm territories and thus, the relative extent of these tissue layers in late embryos. GSK3beta and probably other downstream components of the Wnt pathway thus mediate patterning both along the primary AV axis of the sea urchin embryo and along the dorsal-ventral axis in Xenopus, suggesting a conserved basis for axial patterning between invertebrate and vertebrate in deuterostomes.     

"BEP" RNAs and proteins are situated in the animal side of sea urchin unfertilized egg, which can be recognized by female pronuclear localization

Microsurgery experiments demonstrate that the animal side of the unfertilized sea urchin Paracentrotus lividus egg coincides with the side of the egg pronucleus location. It is demonstrated by means of in situ hybridization and immunostaining of whole mounts of animal or vegetal halves that the previously identified bep 1 and bep4 RNAs and their proteins are located in the animal part of the unfertilized egg and much less in the vegetal part. The addition of Fabs against BEP1 and BEP4 causes exogastrulation.     

beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal-vegetal axis in sea urchin embryos are largely unknown. Nuclear beta-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that beta-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that beta-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal-vegetal axis. Our results also reveal similarities between the sea urchin animal-vegetal axis and the vertebrate dorsal-ventral axis, suggesting that these axes share a common evolutionary origin.     

Characterization of the zebrafish Orb/CPEB-related RNA binding protein and localization of maternal components in the zebrafish oocyte

The animal/vegetal axis of the zebrafish egg is established during oogenesis, but the molecular factors responsible for its specification are unknown. As a first step towards the identification of such factors, we present here the first demonstration of asymmetrically distributed maternal mRNAs in the zebrafish oocyte. To date, we have distinguished three classes of mRNAs, characterized by the stage of oocyte maturation at which they concentrate to the future animal pole. We have further characterized one of these mRNAs, zorba, which encodes a homologue of the Drosophila Orb and Xenopus CPEB RNA-binding proteins. Zorba belongs to the group of earliest mRNAs to localize at the animal pole, where it becomes restricted to a tight subcortical crescent at stage III of oogenesis. We show that this localization is independent of microtubules and microfilaments, and that the distribution of Zorba protein parallels that of its mRNA.     

Differentiation and positioning of nuclei in eggs of the cecidomyid Heteropeza pygmaea

During the first three cleavage divisions of the egg nuclei a precise sequence of spindle orientation and elongation parallel to the longitudinal axis of the egg is apparently involved in positioning one nucleus among the polar granules at the posterior pole of the egg. The size of this nucleus, and the position at which the egg cleaves when pole cell formation occurs, appear to constitute part of the mechanism which ensures that only one nucleus is included in the first pole cell. Blastoderm formation occurs without a well-defined migration of nuclei to the egg surface. Nuclei are so large in relation to the size of the egg that uniform spacing and distribution of nuclei ensures that a large proportion are situated near the egg surface. Those nuclei which are near the egg surface divide synchronously to form a layer of blastoderm nuclei, while membranous cleavage furrows invaginate from the egg surface between them. Nuclei in the central region of the egg chamber condense to form yolk nuclei before blastoderm nuclei have been separated from the rest of the egg by the completion of the cleavage membranes. Polar granules provide the only evidence of fine-structural differences in different regions of the egg chamber cytoplasm. They are found near the posterior pole of the egg from an early stage of oogenesis. They undergo a specific sequence of structural changes and increase in size as the egg grows. No microtubular or microfibrillar arrays have been found in the egg chamber which might form a cytoskeletal basis for spindle orientation or for the spatial differences which develop during differentiation of the uncleaved egg cytoplasm.     

Localization of mitochondrial large ribosomal RNA in germ plasm of Xenopus embryos

In Xenopus, factors with the ability to establish the germ line are localized in the vegetal pole cytoplasm, or germ plasm, of the early embryo [1-3]. The germ plasm of Xenopus, and of many other animal species including Drosophila, contains electron-dense germinal granules which may be essential for germ-line formation [4-5]. Several components of the germinal granules have so far been identified in Drosophila [6-10]. One of these is mitochondrial large ribosomal RNA (mtlrRNA), which is present in the germinal granules (polar granules) during the cleavage stage until the formation of the germ-line progenitors or pole cells [8-9]. MtlrRNA has been identified as a factor that induces pole cells in embryos that have been sterilized by ultraviolet radiation [11]. The reduction of mtlrRNA in germ plasm by injecting anti-mtlrRNA ribozymes into embryos leads to the inability of these embryos to form pole cells [12]. These observations clearly show that mtlrRNA is essential for pole cell formation in Drosophila. Here, we report that mtlrRNA is enriched in germ plasm of Xenopus embryos from the four-cell stage to the blastula. Furthermore, our electron microscopic studies show that this mtlrRNA is present in the germinal granules during these stages. Thus, mtlrRNA is a common component of germinal granules in Drosophila and Xenopus, suggesting that the mtlrRNA has a role in germ-line development across phylogenetic boundaries.     

A review of major nursing vocabularies and the extent to which they have the characteristics required for implementation in computer-based systems

Fertilised mouse eggs develop the oolemma block to sperm penetration within 1 h. This block makes zona-free eggs at the pronuclear stage (zygotes) fully resistant to sperm penetration. In this study we investigated whether this block can spread--as a result of cell fusion--to the oolemma of eggs that are competent to be penetrated by spermatozoa. Preovulatory (GV) oocytes, ovulated oocytes in metaphase II (MII) and 1-cell parthenotes were fused with zygotes and the hybrid cells inseminated at various intervals after fusion. Sperm penetration was assessed on the basis of the presence of Giemsa-positive sperm heads in the air-dried preparations. The oolemma block to sperm penetration develops in all types of hybrids although at different speeds: it develops fast (2-3 h) in oolemma derived from MII oocytes and artificially activated eggs, and slowly in oolemma derived from GV oocytes. In the GV oocyte-zygote hybrids the time of formation of the block varied: while 50% of cells lost the ability to fuse with sperm by 2 h after fusion, in the remaining cells the block must have developed some time between 5 and 18 h after fusion. How these sperm-induced modifications of the oolemma of fertilised egg spread in the hybrid cell and render the 'virgin' part of oolemma resistant to sperm penetration remains unknown.     

Effect of dry fibrin sealant dressings versus gauze packing on blood loss in grade V liver injuries in resuscitated swine

In mammalian fertilization, inositol 1,4,5-trisphosphate receptor (IP3R)-dependent Ca2+ release is a crucial signaling event that originates from the vicinity of sperm-egg interaction and spreads as a wave throughout the egg cytoplasm. While it is known that Ca2+ is released by the type 1 IP3R in the egg cortex, the potential involvement of other isoform types responsible for the Ca2+ rise in the mouse egg (interior) and their spatial distribution are not known. In addition, the biochemical basis has not been definitively established for the development of increased sensitivity to inositol 1,4,5-trisphosphate (IP3) during meiotic maturation. Using specific antibodies to the type 1, 2, and 3 IP3R, we tested the hypotheses that different IP3R isoforms are responsible for the internal Ca2+ elevation and that they contribute to the maturation-associated acquisition of IP3 sensitivity. In both preovulatory oocytes and ovulated eggs of CF-1 mice, immunofluorescence revealed that types 1 and 2 isoforms were present in the cell cortex and interior. Type 1 was observed throughout the cytoplasm, and Western analysis indicated a 1.9-fold maturation-associated increase. In contrast, the signals detected for the type 2 (high-affinity) isoform and type 3 were present to a lesser extent, with type 2 restricted to isolated islands (similar to aggregates of vesicles detected by electron microscopy), which, in the cortex, may amplify early sperm-egg signaling events. The cortical-to-perinuclear localization of the receptor and cortical vesicle aggregates imply an efficient mechanism for propagating Ca2+ release from the cortex into the interior of the egg to activate development, and the isoform localization analysis indicates a clear spatial and biochemical heterogeneity. Types 1 and 2 isoforms were also present in granulosa cells.     

Propagated and nonpropagated calcium transients during egg activation in the annelid, Chaetopterus

Transient waves of Ca2+ release cross-fertilizing deuterostome eggs from the point of sperm entry to its antipode and provide much of the activating stimulus for the egg. Based on several indirect lines of experimental evidence, it was proposed that protostome eggs are activated by a prolonged uptake of Ca2+ from the medium due to sperm-induced membrane depolarization and that this uptake then starts an activation wave similar to those in deuterostomes, except that it moves inward from the whole surface rather than through the egg from pole to pole. To test these hypotheses, we microinjected oocytes of the polychaete annelid, Chaetopterus pergamentaceus, with semisynthetic recombinant aequorins and measured light emission in response to both fertilization and artificial activation by excess K+. Both fertilization and K(+)-activation induced multiple, brief Ca2+ transients in the eggs. The first transient did not propagate, but it was followed by a series of globally propagated Ca2+ waves interspersed with additional nonpropagated pulses. The waves traversed the egg at about 30 micrometer/sec. Sequential propagated waves and nonpropagated pulses generally originated at different regions of the egg surface, except the last few, which originated in the same "pacemaker" region. These new data are consistent with the hypothesis that the activation of protostome eggs is initiated by Ca2+ waves. However, the fact that these waves propagated from pole to pole like those in deuterostome eggs refutes the notion that Ca2+ waves in activating protostome eggs move inward from the whole surface.     

Detection of endothelin-1 and its receptors in rat liver endothelial cells by in situ reverse transcriptase-polymerase chain reaction

Bep mRNAs are localized at the animal pole of P. lividus eggs. In the present communication the secondary structures of the 3'UTRs of the bep1, bep3 and bep4 mRNAs are reported. The minimal lengths of these regions required to bind the 54-kDa protein, previously shown to be involved in localization and anchoring of these RNAs, is estimated. Microinjection of the bep3 3'UTR into egg shows that this RNA fragment is also able to become localized to one of the egg poles, as happens for the entire bep3 RNA.     

Stability in extraversion and aspects of social support at midlife

Fusion of sperm and egg plasma membranes is an early and essential event at fertilization but it is not known if it plays a part in the signal transduction mechanism that leads to the oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]i) that accompany mammalian egg activation. We have used two independent fluorescence methods and confocal microscopy to show that cytoplasmic continuity of egg and sperm precedes the onset of the first [Ca2+]i increase in mouse eggs. The Ca2+ indicator dye Ca2+-green dextran was microinjected and its transfer from egg to sperm was monitored. We found that it occurred before, and without a requirement for, any detectable [Ca2+]i increase in the egg. In separate experiments [Ca2+]i changes were recorded in populations of eggs, using fura red, and the eggs fixed at various times after some of the eggs had shown a [Ca2+]i transient. Fusion of the sperm and egg was then assessed by Hoechst dye transfer. All eggs that showed a [Ca2+]i increase had a fused sperm but more than half of the eggs contained a sperm but had not undergone a [Ca2+]i increase. These data indicate that sperm-egg fusion precedes [Ca2+]i changes and we estimate that the elapsed time between sperm-egg fusion and the onset of the [Ca2+li oscillations is 1-3 minutes. Finally, sperm-egg fusion was prevented by using low pH medium which reversibly prevented [Ca2+]i oscillations in eggs that had been inseminated. This was not due to disruption of signalling mechanisms, since [Ca2+]i changes still occurred if low pH was applied after the onset of oscillations at fertilization. [Ca2+]i changes also occurred in eggs in low pH in response to the muscarinic agonist carbachol. These data are consistent with the idea that the [Ca2+]i signals that occur in mammalian eggs at fertilization are initiated by events that are closely coupled to the fusion of the sperm and egg membranes.     

Viable offspring derived from fetal and adult mammalian cells

Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.