The Periodic Changes in Microvilli Density in Activated Xenopus Eggs That Correspond to the Cleavage Cycle

In order to obtain the cytological basis for the periodic flattening and rounding-up of activated amphibian eggs, the surface ultrastructure and the cortical microfilament organization were studied in Xenopus laevis . Scanning electron microscopy (SEM) of the egg surface revealed that the density of microvilli at the animal pole region decreased significantly when the periodic flattening started, but increased again concomitantly with the commencement of the rounding-up. Isolated pieces of the cortices stained with rhodamine-phalloidin exhibited the periodic disorganization and reorganization of a meshwork with bright dots probably corresponding to microvilli, in good synchrony with the decrease and increase of the microvilli density. Study of appropriate batches of eggs in which the moving front of surface contraction waves (SCWs; 1) can be localized revealed that the decrease and increase of the microvilli density correspond to SCW-1 and -2, respectively. SEM and the cytochemical examination of the eggs from which the germinal vesicle (GV) had been removed revealed that none of these changes occurred in the enucleated eggs. These observations suggest that the GV-dependent regulation of the microfilament organization in an egg cortex constitutes the cytological basis for the SCWs and for the periodic flattening and rounding-up of denuded eggs.     

Cycling of intracellular pH during cell division of Xenopus embryos is a cytoplasmic activity depending on protein synthesis and phosphorylation

In Xenopus embryos, the successive and rapid cell divisions that follow fertilization are accompanied by periodic oscillations of intracellular pH (pHi). Cycling of pHi occurs in phase with several other oscillatory activities, namely nuclear divisions, M phase-promoting factor (MPF) activity, and surface contraction waves (SCWs). We report that treatments that abolish cycling of MPF activity and the SCWs also suppress the pHi oscillations, whereas those that block cell division without affecting neither MPF activity nor the SCWs do not suppress the pHi oscillations. Experiments on enucleated oocytes, matured in vitro and activated, demonstrated that the activity governing the rhythmicity of the pHi oscillations resided in the cytoplasm of the oocyte. In this respect, the activity responsible for the pHi oscillations was different from that which drives the SCWs, which necessitated the presence of the oocyte germinal vesicle (Ohsumi et al., 1986), but more closely resembled MPF activity that did not require the presence of the oocyte germinal vesicle (Dabauvalle et al., 1988). In mature eggs enucleated at the time of egg activation, the pHi oscillations were similar to those in control nucleated eggs, whereas the period between two peaks of SCWs was 35-60 min vs. 20-35 min in nucleated control eggs. Previous studies had shown that the periodicity of SCWs was larger in anucleate egg fragments than in their nucleate counterparts (Sakai and Kubota, 1981), the difference being on the order of 6-15 min (Shinagawa, 1983). However, in these previous studies, enucleation was performed 30-50 min after fertilization. Our results clearly demonstrate that the periodicity of the SCWs is lengthened when the interval between egg activation and enucleation is shortened, thereby providing an easier way to assess the nuclear dependency of the SCWs. Finally, the various possibilities concerning the role of pHi cycling during cell division are discussed.     

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.     

Relative Timing of Stiffening with Various Combinations of Nucleate and Enucleated Egg Fragments of Xenopus laevis

Previously, I found that in Xenopus eggs, the surface contraction wave (SCW) can arise at a point close to a male, a female or a zygote nucleus or a top portion of the egg (9). This finding suggested that all types of nucleus and cytoplasm of a Xenopus egg have the potential to determine the point of initiation of the SCW. Since stiffening is closely associated with the SCW, to determine the factors governing this phenomenon, I compared the times of egg stiffening of pairs of fragments containing different types of nuclei or cytoplasm. I found that the stiffening occurs earlier in fragments containing any type of nucleus than in those containing no nucleus, and earlier in fragments containing a male nucleus than in those containing a female nucleus. These results are consistent with the notion that either a nucleus or cytoplasm that induces the SCW earliest determines the point of initiation of the SCW close to itself. I also found that DNA replication is essential for the earlier occurrence of stiffening in a fragment containing a nucleus.     

Role of nuclear material in the early cell cycle of Xenopus embryos

Activated Xenopus eggs show periodic surface contraction waves and oscillations in endogenous protein phosphorylation, MPF, and kinase activities timed with the cleavage cycle of control fertilized eggs. In this paper, we show that in activated eggs lacking the material that originates from the oocyte nucleus, MPF and kinase oscillations occur in the absence of surface contraction waves. Two mitotic phosphoproteins (M116 and M46), previously described by 32P labeling in nucleated eggs, are no longer detected in the enucleated eggs. We conclude that a cytoplasmic temporal control of MPF and kinase activities is likely to be the essential cell cycle oscillator. The oocyte nuclear components normally stored in the cytoplasm of the embryos are not involved in the clock although they appear to be required for the generation of surface contraction waves.     

Cyclic changes in amphibian egg microvilli occur during the division cycle: implication of MPF.

The microvilli (MV) of Pleurodeles (amphibian) eggs were examined following fertilization and compared with those of artificially activated eggs and enucleated eggs using scanning and transmission electron microscopy. The MV pattern in fertilized eggs was found to undergo a cyclic transformation during the course of the first few division cycles. Similar changes also occurred in the MV of artificially activated eggs and enucleated eggs. The reorganization of the MV was sensitive to cycloheximide and cytochalasin B, but was unaffected by colchicine. Thus, this MV alteration requires protein synthesis and microfilaments but microtubules are not implicated in this process. In addition, the effects on the MV pattern of the maturation or mitosis promoting factor (MPF) were tested. Injection of MPF into eggs at different times during the first division cycle nearly always induced an elongation of the MV. This observation suggests that MPF could regulate either directly or indirectly, via a MPF-sensitive factor, the cyclic transformation of amphibian egg MV.     

Localised MPF regulation in eggs

In this review we discuss the evidence that activation and inactivation of M-phase promoting factor (MPF), the universal mitotic activator, are regulated locally within the cell, and consider the mechanisms that might be responsible. Localised initiation of MPF activation has been demonstrated in Xenopus eggs and egg fragments by examination of the timing of surface contraction waves (SCWs), indicators of MPF activity, and confirmed by direct measurement of MPF in such fragments. Both the timing and the site of SCW initiation relate to the presence of nuclei and of associated centriole-nucleated microtubules. Localised MPF activation is likely to occur in the perinuclear cytoplasm as well as within the nucleus. Studies in a number of cell types show that the perinuclear/centrosomal region is the site of accumulation of MPF itself (the cyclin B-Cdc2 kinase complex) and of many of its molecular regulators. It also harbours calcium-regulating machinery, and in sea urchin eggs is the site of transient calcium release at the onset of mitosis. During mitosis MPF, regulatory molecules and calcium signalling components associate with spindle structures. Inactivation of MPF to end mitosis has been shown to be initiated locally at the mitoic spindle in Drosophila embryos. In sea urchin and frog eggs, calcium transients are required for both mitotic entry and exit and in mouse eggs, MPF inactivation requires both a calcium signal and an intact spindle. It thus appears that calcium signals coinciding with localised accumulation of MPF regulators are required first to set off and/or amplify the MPF activation process around the nucleus, and later to promote MPF inactivation via cyclin B destruction. Calcium release from sequestering machinery organised around nuclear and astral structures may act co-operatively with localised MPF regulatory molecules to trigger both mitotic entry and exit.     

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.     

Spermatozoan response to the toad egg matured after removal of germinal vesicle.

The germinal vesicle (GV) was removed from toad oocytes at various times after treatment with progesterone, and enucleated eggs were inseminated under conditions that ensured fertilization of nucleated control eggs. The eggs enucleated before the initiation of GV break-down did not show genuine cleavage. Cytological examinations revealed, however, that spermatozoa enter the eggs enucleated even before the hormone treatment, without subsequent formation of pronuclei or DNA synthesis. The same lack of response was observed when several detergent-pretreated sperm were injected into enucleated eggs. When GV material was injected back into enucleated oocytes, the injected spermatozoa underwent transformation and DNA synthesis, although in variable degrees, in the egg cytoplasm. It is concluded that the egg becomes fertilizable independently of the GV during the hormone-induced maturation process. After entering the egg, however, spermatozoa require GV material for their participation in the developmental process.     

An intermediate state of fertilization involved in internalization of sperm components

The pathway of sperm entry during sea urchin fertilization was analyzed by using sperm covalently labeled with fluorescent and radioactive tracers. Sperm that have been covalently labeled on their surfaces with fluorescein isothiocyanate (FITC) or a radioactive congener, diiodofluorescein isothiocyanate (¹²⁵IFC), transfer labeled components to the egg that persist throughout early development. In order to study the transfer of sperm components and their fate after fertilization, cytochalasin B-dependent inhibition of fertilization, previously shown to permit the cortical reaction of sea urchin eggs but block sperm pronuclear incorporation, was investigated. Under certain conditions cytochalasin B or D (CB or CD) results in about half of the activated eggs having both the sperm nucleus and the fluorescently labeled sperm components arrested apparently at the level of the egg plasma membrane. This arrest of internalization was reversed by removal of CB or CD, and the sperm derivatives entered the egg. When sperm were labeled noncovalently with ethidium bromide or rhodamine 123, fluorescence was transferred to the egg in the cytochalasin-inhibited state in a fashion similar to that found in normal fertilization; in both cases the sperm fluorescence disappeared within a few minutes of fertilization, due to the repartitioning of the noncovalent dyes into the egg cytoplasm. It is concluded that cytochalasin arrests fertilization at an intermediate step in which the sperm has fused with the egg to achieve cytoplasmic continuity, but in which the subsequent internalization of sperm components is inhibited. After removal of cytochalasins the fluorescent sperm components move from the egg surface to an internal site, a process that can be monitored by time-lapse video microscopy with an image intensifier to permit extended observations of sperm fluorescence. The cytoplasmic location of labeled sperm components was substantiated by autoradiography of early embryos fertilized with ¹²⁵IFC-labeled sperm; transfer of sperm components to an internal site was seen after fertilization of either sea urchin or mouse eggs. Taken together, the data suggest that the fate of the labeled sperm surface components, as well as that of the sperm nucleus, is to be transferred to the egg cytoplasm, and that this transfer is mediated by the actin-dependent cytoskeleton of the egg.     

Rat eggs normally exhibit a variety of surface phenomena during fertilization

The surface topography of the rat egg was examined during fertilization in vitro and in vivo. Using phase optics, 348 in vitro fertilized and 50 in vivo fertilized eggs were continuously monitored throughout the 7-hour period of sperm incorporation. A myriad of different surface configurations were seen, with each egg exhibiting one or more of the following changes. A small number of eggs (4–6%) formed surface elevations over the sperm head after its detachment from the flagellum, 15–30 min after sperm-egg fusion; 1 to 1.5 hr after fusion, 40–50% of the eggs produced the so-called incorporation cone, a prominent surface elevation over the decondensing sperm nucleus. The vast majority of eggs (74–82%) formed surface elevations over the proximal tip of the flagellum 2–3 hr after sperm-egg fusion. These had no association with the decondensing sperm nucleus. A few eggs (11–12%) exhibited multiple protrusions that were distributed randomly about the egg surface, whereas 14–20% did not manifest any surface elevations and remained spherical throughout the sperm incorporation period. Regardless of the type of surface change, all of the eggs resumed a spherical shape by the time sperm incorporation was complete. These observations are in contrast to the conclusions by previous authors that formation of the so-called incorporation cone over the decondensing sperm nucleus is a ubiquitous event.     

Surface contraction waves (SCWs) in the Xenopus egg are required for the localization of the germ plasm and are dependent upon maternal stores of the kinesin-like protein Xklp1

During the first four cell cycles in Xenopus, islands of germ plasm, initially distributed throughout the vegetal half of the egg cortex, move to the vegetal pole of the egg, fusing with each other as they do so, and form four large cytoplasmic masses. These are inherited by the vegetal cells that will enter the germ line. It has previously been shown that germ plasm islands are embedded in a cortical network of microtubules and that the microtubule motor protein Xklp1 is required for their localization to the vegetal pole [Robb, D., Heasman, J., Raats, J., and Wylie, C. (1996). Cell 87, 823-831]. Here, we show that germ plasm islands fail to localize and fuse in Xklp1-depleted eggs due to the abrogation of the global cytoplasmic movements known as surface contraction waves (SCWs). Thus, SCWs are shown to require a microtubule-based transport system for which Xklp1 is absolutely required, and the SCWs themselves represent a cortical transport system in the egg required for the correct distribution of at least one cytoplasmic determinant of future pattern.     

Absence of furrowing activity following regional cortical tension reduction in sand dollar blastomere and fertilized egg fragment surfaces

The purpose of the present investigation was to test experimentally the possibility that division mechanism establishment at the equator of sand dollar eggs may be a consequence of cortical tension gradients between the equator and the poles. Cytochalasin has been shown to decrease tension at the sea urchin egg surface. The concave ends of cytochalasin D-containing agarose cylinders were held against regions of the surface of Echinarachnius parma blastomeres and enucleated fertilized egg fragments. The ability to interfere with normal furrowing activity was used as a biological indicator of the effectiveness of cytochalasin. When agarose containing 2 microg/mL cytochalasin contacted the equatorial region of the blastomeres resulting from the first cleavage, or the equatorial surfaces of nucleated fertilized egg halves, furrowing was blocked, stalled or delayed, indicating that the concentration of cytochalasin was effective. When the same concentration of cytochalasin was applied to the poles, the cells and nucleated fertilized egg fragments divided in the same way as the controls, indicating that the effectiveness of the cytochalasin did not spread from the poles to the equator and that bisection did not interfere with the division of nucleated fertilized egg fragments. When the same concentration of cytochalasin was applied to diametrically opposed surfaces of enucleated, spherical egg fragments, there was no evidence of furrowing activity between the areas that contacted the cytochalasin or in any other part of the surface. Because of the tension-reducing effect of cytochalasin, a tension gradient existed between the regions affected and unaffected by cytochalasin. The results strongly suggest that establishment of the division mechanism by simple gradients of tension at the surface is unlikely.     

Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation.

Recently, we have purified a Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), from oocytes of Xenopus laevis and found that the enzyme is activated within 1 min following fertilization [Sato et al. (1996) J. Biol. Chem. 271, 13250-13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996) Dev. Biol. 177, 558-567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner independent or upstream of calcium signaling.     

Germinal vesicle components are not required for the cell-cycle oscillator of the early starfish embryo

We show that certain events of the cell cycle can still occur in starfish oocytes or fertilized eggs from which the germinal vesicle (the prominent nucleus of prophase-arrested oocytes) has been removed before the induction of meiotic maturation. Two meiotic asters develop following hormonal induction of meiotic maturation in these enucleated oocytes. The asters then divide to form a transient tetrapolar figure. When enucleated oocytes are fertilized, the sperm centrosome duplicates at the times corresponding to each cleavage in control nucleated embryos. Periodic changes in the organization of the asters and in the morphology of the cell surface also occur in synchrony with controls. Decondensation of the sperm nucleus, spindle formation, and cleavage do not occur when enucleated oocytes are fertilized. Ultimately the number of asters increases to approximately 520 (about 2(9] before the pseudo-embryo arrests and cytolyzes. Fertilized eggs from which both pronuclei but not the sperm aster have been removed undergo nine cleavages and then cease cell division. The cessation of division may be related to the events that cause the midblastula transition after seven cleavages in normal nucleated embryos.     

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.     

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.     

The role of calcium, polyamines and centrosomes in the formation and organization of cleavage furrows in amphibian eggs.

Cleavage furrows of amphibian eggs exhibit characteristic morphological features: the presence of finger-like microvilli (MV) along their outer edges, the formation of furrow walls from new plasma membrane lacking MV, and the subsequent retrieval of this membrane during the infolding of the furrow. A similar structure can be induced, specifically, by certain cytoplasmic components such as centrosomes, polyamines and calcium. Their respective roles in the events associated with the furrowing process have been investigated by injecting these agents into nucleated and enucleated Pleurodeles eggs and evaluating their effects using cytochemical labelling of the egg surface with a biotin-streptavidin system. The injection of polyamines (spermine or spermidine) and in some cases, calcium into enucleated eggs provoked MV elongation and the appearance of newly formed, smooth plasma membrane. In these eggs, this membrane was not incorporated into the furrows, and as a consequence, the blastomeres did not actually separate. In contrast, the injection of centrosomes into enucleated eggs induced both the incorporation and internalization of new membrane, resulting in the formation of furrows and a true cellularization of the eggs, identical to the cleavage process observed in fertilized eggs. The present results provide further evidence that the establishment of the furrow depends on two complementary interacting systems: the contractile elements of the egg cortex which regulate the insertion of new membrane and the mitotic center which is essential for the invagination of the furrow.