A transient array of parallel microtubules in frog eggs: potential tracks for a cytoplasmic rotation that specifies the dorso-ventral axis

The dorsoventral axis of the frog embryo is specified by a rotation of the egg cytoplasm relative to the cortex. When eggs undergoing the cortical/cytoplasmic rotation were examined by immunocytochemistry and electron microscopy, an extensive array of parallel microtubules was found covering the vegetal hemisphere of the egg. The microtubules were 1-3 microns deep from the plasma membrane and were aligned parallel to the direction of rotation. They formed at the start of rotation and disappeared at its completion. Colchicine and uv irradiation, inhibitors of the rotation, prevented the formation of the parallel microtubules. Based on these properties, we suggest that the parallel microtubules serve as tracks for the cortical/cytoplasmic rotation which specifies the dorsoventral axis of the embryo.     

Independence of two microtubule systems in fertilized frog eggs: the sperm aster and the vegetal parallel array

Two microtubule-containing structures are implicated in dorsoventral polarization of the frog egg, and we examined the relationship between them. The sperm aster provides a directional cue for a cortical rotation specifying polarity, and a vegetal cortical array of parallel microtubules is likely part of the rotational machinery. The growing aster has an accumulation of microtubules marking the path of the sperm pronucleus, and its microtubules extend into the egg cortex as well as the cytoplasm. To test whether the vegetal parallel array was an extension of astral cortical growth, fertilized or activated eggs were bisected into animal and vegetal fragments. The vegetal fragments formed parallel arrays, even when isolated within a few minutes of egg activation. Neither the sperm centrosome nor another microtubule organizing center in the animal half of the egg is required for formation of the parallel array, but some animal half activity is involved in its disappearance. Correspondence to: R.P. Elinson     

Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs.

Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185-197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.     

Two UV-sensitive targets in dorsoanterior specification of frog embryos

Previous work has shown that ultraviolet (UV) irradiation of fertilized frog eggs yields embryos that lack dorsal and anterior structures. The eggs fail to undergo the cortical/cytoplasmic rotation that specifies dorsoventral polarity, and they lack an array of parallel microtubules associated with the rotation. These eggs can be rescued by tilting with respect to gravity, and normal dorsoanterior development occurs. We find here that UV irradiation of Xenopus prophase I oocytes or Rana metaphase I oocytes also causes the dorsoanterior deficient syndrome, but the UV target is different from that in fertilized eggs. Tilting eggs, irradiated as oocytes, with respect to gravity, does not rescue dorsoanterior development, although lithium treatment does. The UV dose required to produce dorsoanterior deficiency for Rana metaphase I oocytes is much less than that for fertilized eggs, and the oocytes can form the array of parallel microtubules and undergo the cortical/cytoplasmic rotation after fertilization. Despite these features of normal development, no dorsoanterior structures form. While the UV target in fertilized eggs is thought to be the parallel microtubules (Elinson & Rowning, 1988; Devl Biol. 128, 185-197), the UV target in the oocytes may be a dorsal determinant.     

Heat-Induced Reversal of Dorsal-Ventral Polarity in Xenopus Eggs

Heat-treatment of fertilized Xenopus laevis eggs at 30°C induced; 1. conspicuous concentration of the pigment toward the sperm entry point (SEP), 2. eccentric first cleavage furrow formation, and 3. reversal of the dorsal-ventral polarity of the embryos. The optimal treatment was for 2.5 min applied at 20 min postfertilization (p.f.). The rotation movement of the Nile-blue stained spots in the vegetal hemisphere of the heated eggs accurately located the future dorsal midline as in untreated embryos (ref. 22). Exposure of eggs to D₂O also reversed the dorsal-ventral polarity of the embryo suggesting that stabilization of microtubules is involved in the dorsal-ventral axis reversal.     

Nuclear Involvement in Localization of the Initiation Site of Surface Contraction Waves in Xenopus Eggs

The initiation site of surface contraction waves (SCWs) was examined in fertilized, parthenogenetically activated and enucleated Xenopus eggs after either rotation through 90° off the vertical axis or injection of colchicine. In enucleated eggs, SCWs always started from a top site of the egg under all conditions examined. In fertilized or activated eggs, SCWs started, depending on the experimental conditions, from either the sperm entry point, the animal pole region located sideward or the top site of the egg. Histological examinations of fertilized and activated eggs revealed that the nucleus was in most cases positioned close to the initiation site of SCWs under various experimental conditions. It is suggested from these results that the egg cytoplasm has an intrinsic capability of causing the surface to generate SCWs, and that the nucleus is generally involved in localizing the initiation site of SCWs in fertilized or activated Xenopus eggs. A possible mechanism for localizing the initiation site of SCWs in Xenopus eggs is proposed.     

The fine structure of the sperm bundles of the coccid,Aspidiotus perniciosus Cumst., and sperm movement

The mature sperm of A. perniciosus are organized into bundles, about 350 μm long by 9–10 μm wide. Each bundle contains 32 sperm enclosed by a common sheath. The sperm contains an elongated ‘central core’, representing nuclear material, surrounded by a spiral microtubular sheath and cytoplasm. The electron-dense nuclear material is localized in the more pointed half of the sperm. The spiral microtubular sheath is composed of 30— 100 microtubules (depending on the cross-sectional level), situated parallel to the longitudinal axis of the sperm. On the basis of this ultrastructural organization, the motility of the sperm and sperm bundle as a whole is discussed. The sperm of A. perniciosus provide strong evidence that the microtubules arranged asymmetrically represent the elements directly involved in sperm motility.     

Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90° before first cleavage

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90° with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35–38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90° rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90°, but the fate of the blastomeres did not simply show a 90° switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position; (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.     

Spermiogenesis and sperm in Mesostoma viaregginum (Plathelminthes, Rhabdocoela): an ultrastructural study to infer sperm orientation

Spermiogenesis in Mesostoma viaregginum begins with the formation of a zone of differentiation containing striated rootlets, two centrioles, and an intercentriolar body in-between. These centrioles generate two parallel free-flagella with the 9+“1” pattern of the Trepaxonemata growing out in opposite directions. Spermatid differentiation is characterised by a 90° latero-ventral rotation of flagella and a subsequent disto-proximal centriolar rotation, with a distal cytoplasmic projection. The former rotation involves the compression of a row of cortical microtubules and allows recognising a flagellar side and an aflagellar side in the late spermatid and in the mature spermatozoon. At the end of the differentiation, centrioles and microtubules lie parallel to the spermatid axis. The disto-proximal centriolar rotation is proposed as a synapomorphy for the Rhabdocoela. The modifications of the intercentriolar body during spermiogenesis and the migration of the nucleus and the centrioles towards the cytoplasmic distal projection are also described. The mature spermatozoon of M. viaregginum is filiform and tapered at both ends and presents many features found in the Rhabdocoela gametes. The nucleus disappears before the flagellar insertion and a density gradient of mitochondria is observed along the sperm axis. The anterior end of the spermatozoon of M. viaregginum is characterised by a tapering capped by a membrane expansion. This study has enabled us to describe precisely the orientation of spermatozoa in the Rhabdocoela in general: the centriolar extremity is proposed as the anterior one for the Rhabdocoela.     

Analysis of microtubule movement on isolated Xenopus egg cortices provides evidence that the cortical rotation involves dynein as well as Kinesin Related Proteins and is regulated by local microtubule polymerisation

In amphibians, the cortical rotation, a translocation of the egg cortex relative to the cytoplasm, specifies the dorsoventral axis. The cortical rotation involves an array of subcortical microtubules whose alignment is mediated by Kinesin-related proteins (KRPs), and stops as M-phase promoting factor (MPF) activation propagates across the egg. To dissect the role of different motor proteins in the cortical rotation and to analyse their regulation, we have developed an open cell assay system involving reactivation of microtubule movement on isolated cortices. Microtubule movements were dependent on ATP and consisted mainly of wriggling and flailing without net displacement, consistent with a tethering of microtubules to the cortex. Reactivated movements were inhibited by anti-KRP and anti-dynein antibodies perfused together but not separately, the KRP antibody alone becoming fixed to the cortex. Neither antibody could inhibit movement in the presence of MPF, indicating that arrest of the cortical rotation is not due to MPF-dependent inhibition of motor molecules. In contrast, D(2)O treatment of live eggs to protect microtubules from progressive depolymerisation prolonged the cortical rotation. We conclude that the cortical rotation probably involves cytoplasmic dynein as well as cortical KRPs and terminates as a result of local MPF-dependent microtubule depolymerisation.     

Ultrastructure of spermiogenesis and the spermatozoon in Castrada cristatispina Papi, 1951 (Platyhelminthes, Rhabdocoela, Typhloplanida)

Spermiogenesis in Castrada cristatispina begins with the formation of a zone of differentiation containing two centrioles with associated striated rootlets and an intercentriolar body between them. The centrioles give rise to two parallel, free flagella of the Trepaxonemata 9 + '1' pattern, growing out in opposite directions. Spermatids undergo a latero-ventral rotation of the flagella and a subsequent disto-proximal rotation of centrioles, and a distal cytoplasmic projection appears. The former rotation involves the compression of a row of microtubules and allows the recognition of a ventral side and a dorsal side. At the end of the differentiation, the centrioles and cortical microtubules lie parallel to the sperm axis. The modifications of the intercentriolar body and the migration of the nucleus and the centrioles toward the distal projection are described. The mature spermatozoon of C. cristatispina is filiform, tapered at both ends and shares several features with the other Rhabdocoela gametes. Nevertheless, the posterior extremity is capped by an electron-dense material. A gradient between mitochondria and dense bodies exists along the sperm axis. This study has enable us a phylogenetic approach of the Rhabdocoela through a comparison of the ultrastructural features of C. cristatispina with the other Rhabdocoela taxa. We propose the disto-proximal rotation of centrioles as a synapomorphy of the Rhabdocoela.     

Cytoskeletal organization in the oocyte,zygote, and early cleaving embryo of the stripe-faced dunnart (Sminthopsis macroura)

Ovulation occurs in Sminthopsis macroura approximately 160 hr after administration of 1.3 IU PMSG, and yields significantly more oocytes than does spontaneous ovulation (P = 0.001). Germinal vesicle (GV)-stage oocytes have a thin cortical rim of microfilaments, which is disrupted by exposure to cytochalasin D. After GV breakdown, the first meiotic spindle forms subcortically and parallel to the oolemma. It rotates during anaphase and telophase to extrude the first polar body. This rotation is associated with a local cortical concentration of microfilaments, which is extruded in the first polar body. The second meiotic spindle is orthogonal to the surface, and extrusion of the second polar body is not associated with obvious local changes in cortical actin, resulting in a polar body containing little polymerized actin. The sites of second polar body emission and sperm entry are always in the half of the oocyte opposite the concentrating yolk mass, and are within 60° of each other in most oocytes. During the concentration and eccentric movement of the yolk, microfilaments condense around it. During yolk expulsion, these microfilaments become continuous with those located subcortically. During early cleavage, the cytocortex of the zygote, but not of the extruded yolk mass, stains heavily for polymerised actin. Multiple sites of pericentriolar material are detectable in the cytoplasm of some secondary unfertilized oocytes which, in the presence of taxol, generate large cytasters and pseudospindle structures. After fertilization, a large aster is formed in association with the sperm entry point and serves as the center of an extensive cytoplasmic network of microtubules which surrounds but does not enter the yolk mass. Taxol treatment generates small cytasters within this meshwork and promotes selective stabilization of some periyolk microtubules opposite to the sperm aster. © 1995 Wiley-Liss, Inc.     

Gravity can induce epinastic bending of isolated leaves

Abstract Isolated leaves of Plectranthus fruticosus were grown in cubic plastic cuvettes, and were supplied via their cut petioles with nutrient solution and indole-3-acetic acid (10^?6m ). Holes bored in the cuvette walls allowed the petioles to be oriented at approximately 60°, 90° or 120° to the vertical. Growth of the leaves initially oriented at angles of 60° and 90°, which simulated the situation in the intact plant, did not result in epinastic bending of the petiole. Inversion of the leaves (adaxial surface of the petiole downwards) and orientation of the adaxial/abaxial surfaces of the horizontal petiole parallel to the gravity vector, however, yielded strong epinastic bending of the petioles. In the latter case, this bending was not in the direction of the gravity vector (evidence for point (iii), below). Furthermore, epinastic bending occurred, when the isolated leaves were rotated on a clinostat (petioles parallel to the rotation axis or inclined to the rotation axis at an angle of 30°; 3 r.p.m.). Since a possible influence of the shoot was excluded, it is concluded that (i) perception and response are restricted to the leaf, (ii) gravity alone is sufficient to induce epinasty, (iii) a gravitropic component of the response can probably be excluded. The clinostat induced epinasty may not have been caused by nullifying the effect of gravity but due to continuous gravistimulation of the leaf.     

Subcortical Rotation and Specification of the Dorsoventral Axis in Newt Eggs

The specification of the dorsoventral axis in naturally polyspermic eggs of the Japanese newt, Cynops pyrrhogaster , was first examined by studies on the spatial relationship between the dorsal midline of the future body plan and the sperm entrance points (SEPs ¹ ). On local insemination, the dorsal blastopore lip was usually found to be formed opposite the SEPs, as in anuran monospermic eggs. Next the movements of the subcortical layer and the cortex were analyzed. "Subcortical rotation" was observed, similar to that of Xenopus laevis eggs with respect to its timing and extent, and its direction was shown to predict the embryonic axis of the eggs. Thus, the dorsoventral axis was concluded to be determined by essentially the same mechanism in the newt as in Xenopus .
Owing to their large size and long first cell cycle, newt eggs appear to be suitable material for study of subcortical rotation, but their behavior is unique in that subcortical rotation occurs in only the vegetal hemisphere so that the subcortical layer stretches in the future dorsal side. Studies on the movement of Nile blue spots suggested that the cytoplasm under the cortex in newt eggs consists of two layers.     

Ultrastructure of spermiogenesis and spermatozoa in Mesocastrada fuhrmanni Voltz, 1898 (Platyhelminthes, Rhabdocoela, Typhloplanoida)

Electron microscopy of the testes of the free-living flatworm Mesocastrada fuhrmanni collected from temporary freshwater ponds shows stages of spermiogenesis that are like other species of the Typhloplanidae. Spermiogenesis in Mesocastrada fuhrmanni is characterized by the presence, in the spermatid, of a differentiation zone underlain by peripheral microtubules and centered on two centrioles with an intercentriolar body. Two flagella of the 9+“1” pattern of the Trepaxonemata grow out in opposite directions from the centrioles. The flagella undergo a latero-ventral rotation, and a subsequent disto-proximal rotation of centrioles occurs in the spermatid. The former rotation involves the compression and the detachment of a row of cortical microtubules, and allows us to recognize a ventral from a dorsal side. Two features are of special interest at the end of differentiation: peripheral cortical microtubules lie parallel to the sperm axis near the anterior tip, but microtubules become twisted (about 40° with reference to the gamete axis) near the posterior extremity; in the same way, the posterior tip of the nucleus is spiralled. As far as we know, these features are observed for the first time in the Typhloplanidae. The pattern of spermiogenesis and the ultrastructural organization of the spermatozoon are compared with the available data on Typhloplanoida and in particular, species of the Typhloplanidae family.     

Gravity influences the position of the dorsoventral axis in medaka fish embryos (Oryzias latipes)

To determine whether gravity influences the plane of bilateral symmetry in medaka embryos, zygotes were placed with their animal-vegetal axis orientated vertically and with their vegetal pole elevated. Then, at regular intervals during the first cell cycle, the zygotes were tilted 90° for about 10 min and subsequently returned to their original orientation. In embryos tilted during the first half of the first cell cycle, the embryonic shield formed on the side that had been lowermost when the zygote was tilted. In embryos that were tilted twice, first in one direction and then in the opposite direction, the embryonic shield formed on the side that was lowermost the first time. When zygotes were centrifuged at 5 g , the embryonic shield formed on the outwardly radial (centrifugal) side of the embryo. The orientation of the array of parallel microtubules in the vegetal pole region was also influenced by tilting or centrifuging zygotes. No correlation was found between the positions of the polar body and the micropyle and the plane of bilateral symmetry. It was concluded that gravity influences both the plane of bilateral symmetry and the orientation of microtubules in the vegetal pole region of medaka embryos.     

Subcortical rotation in Xenopus eggs: an early step in embryonic axis specification

The amphibian egg undergoes a rotation of its subcortical cytoplasm relative to its surface during the first cell cycle. Nile blue spots applied to the egg periphery move with the subcortical cytoplasm and make rotation directly observable (J.-P. Vincent, G.F. Oster, and J. C. Gerhart (1986). Dev. Biol. 113, 484). We have previously shown that the direction of rotation accurately predicts the orientation of the embryonic axis developed by the egg. This suggests an important role for subcortical rotation in axis specification. In this report, we provide two kinds of experimental evidence for the essential role of rotation, and against a role for other concurrent cytoplasmic movements such as the convergence of subcortical cytoplasm toward the sperm entry point in the animal hemisphere. First, dispermic eggs develop only one embryonic axis, which is oriented accurately in line with the direction of the single rotation movement and not with the two convergence foci that form in the animal hemisphere. Rotation probably modifies the vegetal, not animal, hemisphere since axial development is normal in dispermic eggs despite highly altered animal subcortical movement. Second, we show that the amount of rotation correlates with the extent of dorsal development. UV irradiation of the vegetal hemisphere, or cold shock of the egg, inhibits rotation effectively. When there is no rotation, there is no dorsal development. On average within the egg population, increasing amounts of rotation correlate with the increasingly anterior limit of the dorsal structures of the embryonic body axis. However, individual partially inhibited eggs vary greatly in the amount of axis formed following a given amount of movement. Furthermore, the egg normally rotates more than is necessary for the development of a complete axis. These findings suggest that rotation, although essential, does not directly pattern the antero-posterior dimension of the body axis, but triggers a response system which varies from egg to egg in its sensitivity to rotation. This system is artificially sensitized by exposure of the egg to D2O shortly before rotation. We show that D2O-treated eggs produce extensive axes despite very limited rotation, often developing into hyperdorsal embryos. However, like normal eggs, they depend on rotation and cannot form dorsal structures if it is eliminated.     

Structural basis of motility in the microtubular axostyle: implications for cytoplasmic microtubule structure and function

The gross morphology of the protozoan microtubule axostyle of Saccinobaculus ambloaxostylus can now be described in macromolecular detail. The left-handed coil of the axostyle is seen to be dependent upon the asymmetry inherent in the constituent microtubules as expressed by the specific array of linkages between microtubules and by a possible tendency for microtubules to coil into left-handed helices. The laminated sheets of microtubules are not aligned parallel to the long axis of the organelle, but become increasingly tilted off-axis as one descends through the sheets of microtubules from the convex to the concave surface of the axostyle. Fine-structural analysis of the axostyle indicates similarities of the linkages to dynein. The potential loci of the force-generating protein(s) are discussed as well as implications of the axostyle's structure on general microtubule function.