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.     

Evidences for direct involvement of microtubules in cleavage furrow formation in newt eggs

This paper aims at examining the effect of colchicine, a microtubular poison, on the process of furrow formation in whole eggs and egg fragments as well as the process of artificial induction of furrow-like dents, in eggs of the newt, Cynops pyrrhogaster. To apply colchicine locally to eggs, the eggs were slit across or along a furrow in a colchicine solution during first cleavage. When a slit was made across or in front of a growing furrow at the onset of its growth, the furrow quickly ceased growing and often regressed. Cortices containing an entire growing furrow were isolated along with a thin layer of subcortical cytoplasm immediately after the start of the first cleavage. Furrows in the cortices degenerated when the cortices were cultured in a colchicine solution, whereas they continued growing when they were cultured in Holtfreter's saline. Furrow-inducing cytoplasm was injected to a site beneath the cortex in the animal half of the egg during first cleavage. When a small slit was made close to the site of the injection in a colchicine solution, no furrow-like dent was induced. These results imply that microtubules are directly involved in the generation and growth of cleavage furrows.     

Centrosomes and the Scrambled protein coordinate microtubule-independent actin reorganization

In Drosophila syncytial blastoderm embryos, centrosomes specify the position of actin-based interphase caps and mitotic furrows. Mutations in the scrambled locus prevent assembly of mitotic furrows, but do not block actin cap formation. The scrambled gene encodes a protein that localizes to the mitotic furrows and centrosomes. Sced localization, actin reorganization from caps into mitotic furrows, and centrosome-coordinated assembly of actin caps are not blocked by microtubule disruption. Our results indicate that centrosomes may coordinate assembly of cortical actin caps through a microtubule-independent mechanism, and that Scrambled mediates a second microtubule-independent process that drives mitotic furrow assembly.     

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.     

Localized calcium signals along the cleavage furrow of the Xenopus egg are not involved in cytokinesis

It has been proposed that a localized calcium (Ca) signal at the growing end of the cleavage furrow triggers cleavage furrow formation in large eggs. We have examined the possible role of a Ca signal in cleavage furrow formation in the Xenopus laevis egg during the first cleavage. We were able to detect two kinds of Ca waves along the cleavage furrow. However, the Ca waves appeared after cleavage furrow formation in late stages of the first cleavage. In addition, cleavage was not affected by injection of dibromoBAPTA or EGTA into the eggs at a concentration sufficient to suppress the Ca waves. Furthermore, even smaller classes of Ca release such as Ca puffs and Ca blips do not occur at the growing end of the cleavage furrow. These observations demonstrate that localized Ca signals in the cleavage furrow are not involved in cytokinesis. The two Ca waves have unique characteristics. The first wave propagates only in the region of newly inserted membrane along the cleavage furrow. On the other hand, the second wave propagates along the border of new and old membranes, suggesting that this wave might be involved in adhesion between two blastomeres.     

Cleavage and blastoderm formation in normal and experimentally deformed naked eggs of a dipteran insect

Summary The eggs of the gall midgeHeteropeza pygmaea develop parthenogenetically inside of the mother larva. They lack a chorion and remain enveloped by the follicular epithelium. After experimental elimination of the follicular epithelium naked eggs are formed, which reach the blastoderm stage but remain spherical instead of assuming an elongated shape. To analyze this peculiar egg development and the roles of egg shape and envelope during development, the ultrastructure of cleaving normal and naked eggs was investigated. It was shown that the number of elements of Golgi apparatus and endoplasmic reticulum strongly increases during early cleavage. Their association with cleavage furrows and nuclei suggests that these organelles play a dominant role in membrane production. Egg yolk consists of lipids and glycogen, wheareas no proteins are found. Cleaving eggs contain numerous vesicles with lysosomal characteristics, indicating intense autophagic processes. Cleavage furrow formation occurs independently from the positioning of cleavage nuclei. The numerous microtubules, which are associated with cleavage furrows and nuclei and located in the egg periphery, the intercellular bridges, and in the central part of the egg, suggest that the cytoskeleton has an important role in cleavage furrow formation, blastoderm layer establishment, and yolk localization. Since these processes are accurately accomplished in naked spherical eggs, they can be considered as independent of normal egg shape and the follicular epithelium.     

The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

Formation of new plasma membrane during the first cleavage cycle in the egg of Xenopus laevis: An immunocytological study

Polyclonal antibodies (IS1) reacting specifically with plasma membrane proteins of the Xenopus oocyte were used to study the formation of new plasma membrane in cleavage furrows. Membrane precursors were detected in the inner cytoplasm, then under the plasma membrane of the animal hemisphere and finally on the furrow's edges. Cycloheximide and colchicine caused abnormal distribution of stained material. IS1 antibodies conjugated to colloidal gold, were used to examine the local insertion of membrane precursors into the furrow region by electron microscopy. Membrane precursors were only detected in intracytoplasmic vesicles that fused with the plasma membrane at the edges of the furrow walls. Arrays of microtubules may guide membrane precursors to the site of their insertion in the furrow walls.     

Unequal first cleavage in the Tubifex egg: involvement of a monastral mitotic apparatus

The first cleavage in the freshwater oligochaete Tubifex hattai is unequal and meridional, and produces a smaller cell AB and a larger cell CD. This study traces the process of furrow formation, reorganization of cortical F-actin and the assembly of a mitotic apparatus during this unequal division. Cleavage furrow formation consists of two stages: (i) when eggs are viewed from the animal pole, meridionally running furrows emerge at two points of the egg's equator that are 90° apart from each other and approach the egg axis as they deepen; and (ii) at the midpoint between the equator and the egg center, the bottoms of these furrows link to each other on the animal and vegetal surfaces of the egg and form a continuous ring of constriction in a plane parallel to the egg axis. Egg cortices, isolated during the first step and stained with rhodamine-phalloidin, show that the bottoms of recently formed furrows are underlaid by a belt of tightly packed actin bundles (i.e. a contractile arc). The transition to the second stage of furrow formation coincides with the conversion of these actin belts into a continuous ring of F-actin. Whole-mount immunocytochemistry of microtubules reveals that the first cleavage in Tubifex involves an asymmetric mitotic spindle, which initially possesses an aster at one pole but not the other. This ‘monastral’ spindle is located at the egg's center and orients itself perpendicular to the egg axis. During anaphase, astral rays elongate to reach the cell surface, so that the array of astral microtubules in the plane of the egg's equator covers a sector of 270–300°. In contrast, it is not until the transition to telophase that microtubules emanating from the anastral spindle pole approach the cell margin. If eggs are compressed along the egg axis or forced to elongate, they form monastral spindles and divide unequally. In living compressed eggs, mitotic spindles, which are recognizable as bright streaks at the egg's center, appear not to shift their position along the spindle axis during division, suggesting that without eccentric migration of spindles Tubifex eggs are able to divide unequally. These results suggest that mechanisms that translocate the mitotic spindle eccentrically do not operate in Tubifex eggs during the first cell cycle. The mechanisms that generate asymmetry in spindle organization are discussed in the light of the present results.     

Slow calcium waves accompany cytokinesis in medaka fish eggs

Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell. We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at approximately 0.5 micron/s. The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first waves travel within the assembling contractile band, they would indicate local increases of free calcium to concentrations of about five to eight micromolar. This is the first report to visualize high free calcium within cleavage furrows. Moreover, this is also the first report to visualize slow (0.3-1.0 micron/s) as opposed to fast (10-100 microns/s) calcium waves. We suggest that these first waves are needed for furrow growth; that in part they further furrow growth by speeding actomyosin filament shortening, while such shortening in turn acts to mechanically release calcium and thus propagates these waves as well as furrow growth. We also suggest that the second waves act to induce the exocytosis which provides new furrow membrane.     

Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to cleavage furrows and propagated along the furrows as calcium waves. The calcium transients at the cleavage furrows were observed at each cleavage furrow at least until blastula stage. The velocity of the calcium waves at the first cleavage furrow was approximately 3 microns/s, which was much slower than that associated with fertilization/egg activation. These calcium waves traveled only along the cleavage furrows and not in the direction orthogonal to the furrows. These observations imply that there exists an intracellular calcium-releasing activity specifically associated with cleavage furrows. The calcium waves occurred in the absence of extracellular calcium and were inhibited in embryos injected with heparin an inositol 1,4,5-trisphosphate (InsP3) receptor antagonist. These results suggest that InsP3 receptor-mediated calcium mobilization plays an essential role in calcium wave formation at the cleavage furrows.     

Microinjection of Ca2+ store-enriched microsome fractions to dividing newt eggs induces extra-cleavage furrows via inositol 1,4,5-trisphosphate-induced Ca2+ release.

The cleavage signal transferred to the future cleavage cortex during anaphase has been proposed as "cleavage stimulus," but no signal has proved to induce cleavage furrows. The local Ca2+ transient along the cleavage furrow has been reported, but the Ca2+ source has remained unknown. To address these questions, we studied functions of Ca2+ stores in dividing newt eggs and found that microinjection of the Ca2+ store-enriched microsome fraction to the dividing newt egg induced a local extra-cleavage furrow at the injection site in 64-67% of the injected newt eggs while coinjection with inositol 1,4, 5-trisphosphate receptor (IP(3)R) antagonists heparin or anti-type 1-IP(3)R antibody clearly suppressed this induction (5 and 11% in induction rates, respectively). Injection of cerebellar microsomes from the type 1-IP(3)R-deficient mice induced extracleavage furrows albeit at a low rate (19%). Our observations strongly suggest that Ca2+ stores with IP(3)R induce and position a cleavage furrow via IP(3)-induced Ca2+ release (IICR) as Ca(2+)-releasing machinery and putative cleavage stimulus itself.     

Holoblastic early cleavage of Tetrodontophora bielanensis (Collembola) eggs, with special reference to its irregularity.

The fertilized eggs of Tetrodontophora bielanensis start to cleave 6 to 8 days after oviposition and initially only karyokineses occur. The cytokinesis begins after two karyokineses, when four nuclei are observed in the ooplasm. Two cleavage furrows, perpendicular to each other, appear simultaneously at the egg poles where polar bodies are located and gradually the furrows encompass the whole egg diameter. The furrow formation is initiated by the bundle of microfilaments that contract and pull superficial fragments of the oolemma into the yolk and subsequently new membranes, separating the daughter cells, start to form. However, they do not grow towards the egg centre but bifurcate, leaving the central part of the ooplasm outside of the newly formed blastomeres. Starting from the fourth or fifth cleavage division, the bifurcations permanently occur and multiple cleavage furrows are formed on the embryo surface. Moreover, fragments of the ooplasm, enclosed within the cell membrane but devoid of cell nucleus are observed. During further development such cell fragments become reincorporated into the embryo. This mode of cleavage leads eventually to the formation of cellular blastoderm on the embryo surface. The results presented in the paper suggest that the control of cleavage in T. bielanensis acts not at the level of cytoplasmic determinants but rather at the level of positional information of blastomeres.     

The action of cytochalasin B during egg cleavage in Xenopus laevis: dependence on cell membrane permeability

By exposing Xenopus eggs during the first cleavage to cytochalasin B (CCB) for successive periods of 4 min, it has been shown that CCB sensitivity becomes manifest approximately 7 min after the onset of furrow formation. However, even before this time furrow regression can be induced by the injection of CCB under the membrane in the furrow. This shows that during the first 7 min of cleavage the operative contractile system is CCB sensitive. Using microelectrode techniques, electrical membrane characteristics (membrane potential and resistance) were measured continuously in normally cleaving eggs and in cleaving eggs injected with CCB. It was found that the onset of sensitivity to externally applied CCB coincides with a rapid alteration of the membrane potential and resistance. We have concluded that externally applied CCB can only enter the egg when the membrane permeability increases. No evidence has been found that CCB alters the ionic permeability of preexisting cell membrane.     

Cytological and biochemical analyses of the maternal-effect mutant embryos with abnormal cleavage furrow formation in Xenopus laevis

We describe an embryonic lethal mutation in Xenopus laevis that provokes regression of cleavage furrow formation. The mutant females (designated as af) were obtained by the back-cross of a female with one of her sons. All the fertilized eggs laid by the mutant females, regardless of the wild-type male used in the mating, failed to cleave although each furrow ran at a proper position superficially. Light and electron microscopic observations of the embryos revealed that the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions did. Two-dimensional gel-electrophoretic comparisons of af and wild-type embryos demonstrated that two proteins, having estimated molecular masses of about 38 kDa (pI 6.6) and 78 kDa (pI 7.6), were missing in af embryos. Microinjection of clear cytoplasm from a wild-type egg into fertilized af eggs provoked partial surface contraction and cleavage furrow formation in recipient af eggs. The results showed that the af females carry a lethal maternal-effect mutation which causes cleavage furrow regression by being deficient in a few proteins, and that cytoplasm of wild-type eggs can partially rescue the cleavage furrow formation of af eggs by furnishing the corrective material, presumably a product of the normal allele of af.     

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.     

Effects of polyamines on the first division cycle of Xenopus laevis eggs

The involvement of polyamines in cytokinesis has been examined in eggs of the amphibian X. laevis. Microinjection of spermine or spermidine into unfertilized eggs induced a shortening of the first division cycle and early formation of cleavage-like constrictions. Eggs were activated by injection and developed furrows about 45 min later, whereas the first division normally occurred around 120 min after activation. In terms of concentration, spermine was slightly more effective than spermidine, but putrescine had no influence on the division cycle.     

Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs

We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injection of karyoplast nuclei with centrosomes reconstituted the formation of spindle-like structures with well-defined poles. We conclude from these experiments that in mitosis, the centrosome acts as a microtubule-organizing center only in the proximity of the nucleus or chromatin, whereas in interphase it functions independently. The general implications of these results for the interconversion of metaphase and interphase microtubule arrays in all cells are discussed.     

Furrowing in altered cell surfaces.

Understanding the process which established the cell division mechanism requires analysis of the role of the responding surface as well as that of stimulatory subsurface structures. Cell surface was altered by the expansion which occurs during exovate formation. Exovates appear on the surface of fertilized Arbacia lixula, Paracentrotus lividus and Echinarachnius parma eggs in response to extreme flattening. They result from cytoplasmic outflow initiated in a very restricted portion of the egg surface. Observations of the formation process in pigmented A. lixula eggs revealed that the original surface may be expanded about 100 fold as the exovate swells. When exovates formed 15-30 minutes after fertilization contain the mitotic apparatus, they divide synchronously with flattened controls. If nucleated exovates are established after the beginning of first cleavage, furrows appear in ten minutes. Exovates established after the beginning of second cleavage develop furrows four minutes after the entrance of the the mitsotic apparatus. Cytoplasm beneath damaged exovate surfaces sometimes develops partial constrictions independently of the surface in the plane the furrow would have occupied. These results suggest that normal surface structure is unnecessary for furrow establishment and function.     

Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.