Timing system for the start of gastrulation in the Xenopus embryo

This study examined which component of the egg, the nucleus or cytoplasm, is involved in the timing of the start of gastrulation in the Xenopus embryo, and when it starts to measure time. First, nuclei of cells of 256-cell stage embryos were transplanted to enucleated eggs 60 min after activation. These eggs showed first cleavage 20-30 min later than control eggs fertilized at the same time as the activation of recipient eggs, and started gastrulation 25-35 min later than control embryos (depending on the delay in the first cleavage). Second, eggs whose nuclei were temporarily isolated by the extrusion of the portion containing the nucleus out of the fertilization envelope showed first cleavage 60-90 min later than sibling control eggs, because of delayed introduction of the nucleus from the extruded portion. They started gastrulation 60-90 min later than sibling control embryos (depending on the delay in the first cleavage). The portion inside the envelope underwent two to three rounds of oscillation in cell cycle relevant activities before the first cleavage, while the portion outside underwent the same rounds of cleavage as the inside portion. From the present and previous results it is concluded that the putative timing system for the start of gastrulation in the Xenopus embryo, whether it consists of a single or of multiple clocks, starts measuring time at or around the first cleavage, and that the presence of both the nucleus and the cytoplasm in the same cell and occurrence of mitosis and/or cleavage there are indispensable for the timing system to work, although the role of the cytoplasm is superior to that of the nucleus.     

RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.     

Regeneration of sialic acid on the surface of Chinese hamster cells in culture. I. General characteristics of the replacement process

Electropotential differences between the cell interior and the external medium have been studied with intracellular microelectrodes in ovarian oocytes, ovulated unfertilized eggs and fertilized eggs of R. pipiens. In ovarian oocytes the cytoplasm was 50 to 80 mV negative, relative to isotonic Ringer's solution. In contrast, electrode penetration of the oocyte nucleus in situ indicated that the nucleoplasm was about 25 mV positive, relative to the cytoplasm. After ovulation, the cortical cytoplasm became 20 to 50 mV positive with regard to an external solution of 0.1 strength Ringer's solution (ca. pond water). Penetration of the cytoplasm at levels from 0.3 to 0.6 mm below the egg surface revealed an inner zone with a potential which was about 15 mV negative, relative to the cortical cytoplasm. A slow hyperpolarization of the cortical membrane occurred at activation, with the potential returning to that of the ovulated unfertilized egg within ten minutes. After fertilization, the egg cytoplasm remained positive until the first cleavage. As division proceeded, the cytoplasm slowly depolarized and became 50 to 60 mV negative, relative to 0.1 strength Ringer's solution.     

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.     

Developmental Timing in Fertilized Merogones of the Sand Dollar, Clypeaster japonicus

Pairs of nucleate and non-nucleate fragments prepared by manually bisecting unfertilized eggs of Clypeaster japonicus were inseminated to obtain a pair of egg fragments, one with a diploid nucleus (zygote) and the other with a male pronucleus (merogone). Haploid male pronuclei in the merogone always entered the first mitosis (NEB) later than the zygote partner. The delay in NEB ranged from 6 to 24 min in different pairs. The interval from the first NEB to formations of the karyomere and the cleavage furrow, and the durations of the second, third and fourth cell cycles were identical in the zygote-merogone pairs. Observations of Colcemid-treated eggs suggested that the duration of the first DNA synthetic period was prolonged in the merogone. From this observation of wide variation in the time of the first mitosis, in contrast to constant durations of subsequent mitotic intervals, we suppose that some critical event triggers the transition of development for the start of well-ordered sequences in the mitotic cycle in sea urchin development.     

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo.

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.     

Microtubules During Blastoderm Formation in the Egg of the Silkworm, Bombyx mori L.

Microtubules in the silkworm egg, Bombyx mori , were observed by electron microscopy, in order to investigate the relationship between cytoskeletal organelles and the migration of energids, the cleavage nuclei accompanied by the associated cytoplasm, near the egg surface or during blastoderm formation. Numerous microtubules were observed in the associated cytoplasm of an energid even in the interphase of mitosis.
At about 8.5 hr after oviposition, when many energids had already cleft and distributed near the peripheral yolk granule region, long microtubules distributed radially from the perinuclear region to the periphery in the associated cytoplasm. When an energid was protruding, the microtubules above the nucleus distributed at a more acute angle than those under the nucleus. When a blastoderm cell had just been formed, the microtubules were observed only under the nucleus.
Colchicine, an inhibitor of microtubules, stopped the migration of energids and inhibited the formation of blastoderm cells even after many energids had already distributed at the peripheral yolk granule region. The relationship between the microtubules and the migration of energids near the egg surface or during blastoderm formation was discussed.     

Axis establishment and microtubule-mediated waves prior to first cleavage in Beroe ovata

The single axis (oral-aboral) and two planes of symmetry of the ctenophore Beroe ovata become established with respect to the position of zygote nucleus formation and the orientation of first cleavage. Bisection of Beroe eggs at different times revealed that differences in egg organisation are established in relation to the presumptive oral-aboral axis before first cleavage. Lateral fragments produced after but not before the time of first mitosis developed into larvae lacking comb-plates on one side. Time-lapse video demonstrated that waves of cytoplasmic reorganisation spread through the layer of peripheral cytoplasm (ectoplasm) of the egg during the 80 minute period between pronuclear fusion and first cleavage, along the future oral-aboral axis. These waves are manifest as the progressive displacement and dispersal of plaques of accumulated organelles around supernumerary sperm nuclei, and a series of surface movements. Their timing and direction of propagation suggest they may be involved in establishing cytoplasmic differences with respect to the embryonic axis.Inhibitor experiments suggested that the observed cytoplasmic reorganisation involves microtubules. Nocodazole and taxol, which prevent microtubule turnover,blocked plaque dispersal and reduced surface movements.The microfilament-disrupting drug cytochalasin B did not prevent plaque dispersal but induced abnormal surface contractions. We examined changes in microtubule organisation using immunofluorescence on eggs fixed at different times and in live eggs following injection of rhodamine-tubulin. Giant microtubule asters become associated with each male pronucleus after the end of meiosis. Following pronuclear fusion they disappear successively, those nearest the zygote nucleus shrinking first, to establish gradients of aster size within single eggs. Regional differences in microtubule behaviour around the time of mitosis were revealed by brief taxol treatment, which induced the formation of small microtubule asters in the region of the nucleus or spindle during both first and second cell cycles. The observed wave of change may thus reflect the local appearance and spreading of mitotic activity as the zygote nucleus approaches mitosis.     

Cyclic regulation of cytokinesis in amphibian eggs

The effects of amphibian egg cytoplasm extracted at different times after activation and during the first four cleavages on cytokinesis were examined. Extracts of artificially activated or fertilized Xenopus or Pleurodeles eggs taken at the time of activation (T = 0) provoked precocious cleavage furrows in Pleurodeles eggs. Between T = 0.25 and T = 0.75 of the first cell cycle, the period corresponding to interphase, an inhibitory effect was found, and the division of injected eggs was delayed up to 30%. After T = 0.75, that is during mitosis, the cleavage induction effect was observed again. These enhancing and inhibitory effects were also found in the two fractions obtained following gel filtration of the cytoplasmic extracts. These experiments support the hypothesis that two antagonistic factors control cytokinesis. The inhibitory factor is active only during interphase, while the positive factor is present during mitosis and appears to regulate cytokinesis.     

Immunocytochemical evidence for centrosomal phosphoproteins in mitotic sea urchin eggs

The change in distribution of centrosomal phosphoproteins was examined in sea urchin eggs from fertilization to the first cleavage by immunofluorescence staining with the anti-phosphoprotein antibodies, MPM-1 and MPM-2. The antibodies reacted with female pronuclei in unfertilized eggs as well as centriolar complexes located at the base of sperm flagella. After insemination, male and female pronuclei fused together to form a zygotic nucleus which was visualized by staining of fertilized eggs with the antiphosphoprotein antibodies. No major change in staining pattern was detected in extracted whole eggs until mitosis. As the fertilized eggs approached mitosis, however, the antigens started to redistribute from nuclei to the perinuclear position where the mitotic centrosomes were located. Detailed immunofluorescence observation of isolated spindles revealed that the phosphoantigens were retained in isolated structures. A major 225 kd polypeptide was recognized by the antibodies, suggesting that the 225 kd protein is a phosphocomponent of centrosomes. The area recognized by the antibody in mitotic poles enlarged with the progress of mitosis, suggesting that the antigens were apparently localized in the centrosphere. Centrospheres prepared from isolated spindles by salt extraction strongly reacted with the antibodies. One or two bright dots, which may represent centrioles, were visible in the isolated centrosphere. At the end of mitosis, the antigens again appeared in the newly formed daughter nuclei. Centriole-containing cytasters and centriole-free monasters were parthenogenetically induced in unfertilized eggs (Kuriyama and Borisy, (1983) J. Cell Sci. 61: 175-189). The antibodies stained centers of both the asters whether they contained centrioles or not, indicating that the antibodies recognizes the components of the pericentriolar material.     

Intracellular distribution of calcium and phosphorus during the first cell division of the sea urchin egg

The intracellular distribution of calcium and phosphorus during metaphase and anaphase of the first cleavage in sea urchin eggs was studied with the electron-probe microanalyzer. This study allowed a comparison of the relative concentrations of both elements on the polar and cleavage furrow regions of the membrane and on the mitotic asters and cytoplasm. The results show that in most eggs, both calcium and phosphorus are more highly concentrated in the mitotic asters than in surrounding cytoplasm during both anaphase and metaphase. Calcium is more concentrated at the furrow region than at the polar region during metaphase but not anaphase. The role of calcium during mitosis was reviewed with special reference to the theories on the formation of the cleavage furrow along the equatorial zone between two mitotic centers.     

Studies on cyclic AMP levels and phosphodiesterase activity in developing sea urchin eggs : Effects of puromycin, 6-dimethylamino purine and aminophylline

Cyclic adenosine monophosphate (CAMP) was measured in sea urchin eggs by the binding assay method of Gilman and with a radioimmune assay procedure. Intracellular concentrations of the nucleotide in unfertilized eggs were about 1.5 × 10⁻⁷ M and rose to about 3 times this value at first cleavage. Aminophylline, a known inhibitor of phosphodiesterase was shown to cause an increase in intracellular levels of CAMP by first cleavage and to inhibit phosphodiesterase activity in homogenates of both unfertilized and fertilized eggs. Puromycin and its purine component, 6-dimethylaminopurine (DMAP), did not cause an increase in intracellular CAMP levels and did not inhibit phosphodiesterase activity at concentrations an order of magnitude higher than those at which they inhibit cell division.     

Induced formation of asters and cleavage furrows in oocytes of Xenopus laevis during in vitro maturation

We have previously reported that injection of purified basal bodies or sperm into unfertilized eggs of Xenopus laevis induced the formation of asters and irregular cleavage furrows. Fully grown oocytes were found to be unable to form asters or cleavage furrows. In this paper we show that the oocyte acquires the ability to form asters upon basal body injection at the time of germinal vesicle breakdown during in vitro maturation. Our evidence indicates that aster formation requires progesterone-stimulated changes in the oocyte and mixing of cytoplasm and germinal vesicle plasm. The ability of the oocyte to form cleavage furrows arises six to eight hours after germinal vesicle breakdown. We infer that some maturational change in the cell cortex occurs to enable the egg surface to furrow. Experiments on the relationship of aster formation to furrow initiation indicates that asters stimulate furrow formation. However, some furrowing could be induced without aster formation in mature oocytes and unfertilized eggs by an activation stimulus, showing that asters are not essential for cleavage initiation. The significance of these observations are discussed in the light of our current understanding of meiotic maturation, cell cleavage and aster growth.     

THE CYTOPLASMIC CONTROL OF NUCLEAR ACTIVITY IN ANIMAL DEVELOPMENT

1.This article reviews the occurrence, mechanism, and functional significance of the cytoplasmic regulation of nuclear activity during cell differentiation and especially during early animal development. 2.Nuclei from brain, and from other kinds of adult cell normally inactive in DNA synthesis, are rapidly induced to commence DNA synthesis by components or properties of intact egg cytoplasm. The components of egg cytoplasm which induce DNA synthesis are not species-specific and they are likely to include DNA polymerase. It is known that DNA polymerase exists in egg cytoplasm before it becomes associated with nuclei in which it is effective. The induction of DNA synthesis in brain nuclei by living egg cytoplasm is always preceded by a pronounced nuclear swelling, a dispersion of chromosomes or chromatin, and the entry of cytoplasmic protein into the nucleus. 3.RNA synthesis can be experimentally induced or repressed by living cytoplasm. The cytoplasm of unfertilized and fertilized eggs appears to contain components which can reversibly and independently repress the synthesis of ribosomal RNA, transfer RNA, and heterogeneous RNA. RNA synthesis can be induced by introducing nuclei inactive in this respect into the cytoplasm of cells very active in RNA synthesis. The induction and repression of RNA synthesis is preceded by a marked swelling of the nucleus and the dispersion of its chromosome material. 4.The cytoplasmic control of chromosome condensation before division has been demonstrated by introducing sperm or adult brain nuclei into the cytoplasm of oocytes undergoing meiotic maturation. 5.The evidence that regional differences in the composition of eggs and other cells are associated with changes in nuclear and gene activity is reviewed in Section 111. While it is certain that these regional differences are of great importance in cell differentiation, evidence that they have a direct effect on nuclear activity has been obtained in a few instances only. In some species it has been shown that the cytoplasmic components related to germ-cell differentiation include RNA and, frequently, granules. 6.It is concluded that whenever nuclei are introduced experimentally into the cytoplasm of another cell, they very quickly assume, in nearly every respect, the nuclear activity characteristic of the host cell. In many instances, altered function has been demonstrated in nuclei which subsequently support normal development. The induced nuclear changes are therefore regarded as normal and it is believed that they are achieved through the same mechanism as that by which the host cell nucleus originally came to function in its characteristic way. Examples are cited to show that changes in gene activity very frequently arise immediately after mitosis. The changes induced experimentally in transplanted nuclei resemble in very many respects those undergone by nuclei which are naturally reconstituted after mitosis, and it is argued that the two processes are functionally equivalent, It is suggested that during telophase of mitosis, chromosomes are reprogrammed in respect of potential gene activity by association with cytoplasmic proteins. Inter-phase nuclei seem not to show changes of gene activity except when they undergo a pronounced enlargement after entering a new cytoplasmic environment.     

Hypotonic buffer induces meiosis and formation of anucleate cytoplasmic islands in the egg of the two-spotted cricket Gryllus bimaculatus

In insects, egg activation is known to occur in vivo and independently of fertilization, but its mechanisms are poorly understood. To gain understanding of these mechanisms, an attempt was made to activate the egg of Gryllus bimaculatus in vitro. It was found that meiosis resumed and was completed in unfertilized eggs treated with hypotonic buffer. Early developmental processes in activated, unfertilized eggs were investigated and compared with those in fertilized eggs. Mitosis did not progress, resulting in formation of anucleate cytoplasmic islands (pseudoenergids). Development in the activated, unfertilized eggs stopped at this stage and both yolk subdivision and cellularization did not occur. To elucidate the role of the nucleus in the developmental process to the syncytial stage in fertilized eggs, eggs were treated with aphidicolin to inhibit DNA polymerization. It was found that pseudoenergids also formed in these aphidicolin-treated fertilized eggs. These results demonstrate that pseudoenergids can increase in number independently of nuclei, suggesting that the cytoplasm rather than the nucleus plays the primary role in development to the syncytial stage in G. bimaculatus.     

Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.     

Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 microM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca(2+) ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca(2+) was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca(2+).     

Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development

Phospholipase C-zeta (PLCzeta), a strong candidate of the egg-activating sperm factor, causes intracellular Ca2+ oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLCzeta. Changes in the localization of expressed PLCzeta were investigated by tagging with a fluorescent protein. PLCzeta began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLCzeta in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLCzeta was recognized in every embryo up to blastocyst. Thus, PLCzeta exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca2+ oscillations in early embryogenesis.     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.