Oogenesis in a paedogenetic dipteran insect under normal conditions and after experimental elimination of the follicular epithelium

Summary Paedogenetically developing eggs of the gall midgeHeteropeza pygmaea are not deposited, but develop in the hemocoel of the mother larva. The nurse chamber remains present in the cleaving egg, and the follicular epithelium does not form a chorion but envelops the growing egg during embryonic development. It is possible to obtain naked eggs, i.e. eggs lacking the follicular epithelium, which are able to develop up to the blastoderm stage but remain spherical instead of assuming an elongated shape. Oogenesis of normal and naked eggs has been studied at the ultrastructural level with special reference to the nurse chamber. It is shown that the nurse chamber nuclei develop large nucleoli during oogenesis, indicating that the nurse chamber supplies the oocyte with ribosomal RNA (rRNA). The dense bodies in the nurse chamber may represent an intermediate stage in the transport of the rRNA from the nurse chamber to the oocyte; they are probably not related to the polar granules in the oocyte. It is also shown that the intercellular bridge joining the nurse chamber to the oocyte disappears shortly before cleavage initiation. During egg cleavage the follicular epithelium surrounds the nurse chamber, which degenerates and is gradually absorbed by the growing egg plasmodium. Naked cleaving eggs are never attached to a nurse chamber or to relics of it. Naked oocytenurse chamber complexes frequently aggregate, which may indicate a role of the follicular epithelium in follicle separation during normal development.     

Hsüa deflexa sp. nov. from the Xujiachong Formation (Lower Devonian) of eastern Yunnan, China

A new vascular plant, Hsüa deflexa sp. nov., is documented from the Lower Devonian ((upper) Pragian-lower Emsian) Xujiachong Formation, near Xujiachong village, Qujing District, eastern Yunnan, South China. In three dimensions, the branching system comprises a robust creeping main axis and comparatively slender erect lateral axes inserted oppositely or alternately. The lateral axes depart at right angles from the main axis. Towards the apex, the main axis is perhaps erect with the lateral axes attached at smaller angles. The lateral axes dichotomize equally one to three times in different planes and decrease in diameter and length acropetally. They bear sparse and irregular tiny spines. Apices of a pair of distal lateral axes curve in opposite directions and each terminates in a rounded to reniform sporangium. The sporangium dehisces into two equal valves along an indistinct convex marginal thickening. The xylem is possibly centrarch. This plant fits Hsüa in branching pattern, sporangial characters and xylem maturation. Hsüa deflexa sp. nov. differs mainly from the type species H.  robusta in the presence of axial spines, perpendicular extension of lateral axes from the main axis and curving of distal lateral axes. Based on the possibly centrarch xylem and terminal sporangium, this plant relates to the rhyniophytes ( sensu Banks, 1968). In view of the equal sporangial valves with marginal thickenings it resembles the zosterophyllophytes ( sensu Banks, 1968). Hsüa is now treated as incertae sedis .  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142 , 255−271.     

Effect of ecdysone agonist RH-0345 on reproduction of mealworm,Tenebrio molitor

RH-0345 belongs to a new group of insect growth regulators (IGRs) with a benzoylhydrazine structure that mimic the action of the natural insect molting hormone 20-hydroxyecdysone. After topical application on female adult beetles of mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), first oviposition was delayed, the number of eggs per female was reduced by 32%, the follicular epithelium was thinner (-33%) during sexual maturation, the size of deposited eggs was reduced, and egg viability was lost by 68%. Treatment with RH-0345 had also reduced the ovarian protein content and two protein bands were missing in the ovaries. Ultrastructural observations of the ovaries at the end of vitellogenesis in treated females, however, showed no evident differences with the fine structure of both follicular cells and oocytes in controls. In addition, we measured the amount of ecdysteroids in the medium of treated ovary cultures in vitro and in the eggs deposited by treated females. Possible action sites with the reproductive system at different levels in T. molitor are discussed for this novel group of IGRs.     

Polarity of the ascidian egg cortex before fertilization.

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.     

Cortical differentiation of the animal pole during maturation division in fertilized eggs of Tubifex (Annelida,Oligochaeta): I. Meiotic apparatus formation

The fine structure of the animal pole cortex is examined in the fertilized Tubifex egg undergoing the formation of the second meiotic apparatus (MA). The fully formed MA which orients its axis at right angles to the surface is found at the animal pole about 40 min after formation of the first polar body. It is composed of a spindle and asters at its poles; a centriole is found in the inner aster, but not in the peripheral aster adjacent to the surface. During the formation of the MA, the animal pole surface is lined with a 0.15-μm-thick, electron-dense cortical layer, which is rich in microfilaments. The arrangement of the filaments in the layer changes from a parallel array to a meshwork with progressive formation of the MA. Microtubules of the peripheral aster terminate in the cortical layer. When a jet stream of glycerol/dimethyl sulfoxide solution is applied to an egg fragment glued on a polylysine-coated coverslip, an egg cortex-MA complex is isolated on the coverslip; the MA appears to be tethered to the egg surface by the structural connection between the filamentous cortical layer and microtubules of the peripheral aster. Cytochalasin B (50 μg/ml), when administrated at early phase of the MA formation, does not show any effect on the structure of the cortical layer and the MA; however, if eggs shortly before the termination of the first polar body formation are immersed in the same test solution, the cortical layer of the animal pole becomes thinner, and the filamentous material is not observed in it. Furthermore, in these eggs, the peripheral aster and the spindle are not structurally discernible because of the suppression of microtubule assembly, whereas microtubules on kinetochores and in the inner aster are normally developed. These results are discussed in relation to the role of the animal pole cortex in fixing of the MA to the egg surface and in forming of the MA.     

The role of the follicular epithelium in growing eggs of a dipteran insect during late oogenesis and cleavage

The gall midge Heteropeza pygmaea can reproduce by means of paedogenesis (i.e., larval parthenogenesis). In that process, follicles are produced that develop while floating in the hemocoele of the mother larva. A chorion is not formed at the end of oogenesis, and the growing embryos remain enveloped by the follicular epithelium. To investigate possible adaptations of the follicular epithelium to this unusual egg development, its ultrastructure has been studied during late oogenesis and cleavage. Earlier investigations had shown that the follicle cells are provided with a specifically arranged microtubular frame, which may be responsible for the anisometric growth of the egg. The present work shows that the follicle cells are always joined by desmosomes and septate junctions. During development, the septate junctions increase their surface and change their orientation to become parallel to the longitudinal egg axis, thus increasing the resistance of the follicle cells to being torn apart by growth tensions. The total surface of the follicular epithelium increases during development. Well-developed nucleoli in the nuclei and numerous ribosomes in the cytoplasm of follicle cells indicate a high level of synthetic activity. This activity may be required to support the increase in the membrane surface and the establishment of the microtubular frame. Lipid droplets, glycogen, and different inclusions in the follicle cells may represent nutrient and energy reserves. Structures indicating a quantitative significant transfer of nutrients from the follicle cells to the egg were not found.     

Determination of cell division axes in the early embryogenesis of Caenorhabditis elegans

The establishment of cell division axes was examined in the early embryonic divisions of Caenorhabditis elegans. It has been shown previously that there are two different patterns of cleavage during early embryogenesis. In one set of cells, which undergo predominantly determinative divisions, the division axes are established successively in the same orientation, while division axes in the other set, which divide mainly proliferatively, have an orthogonal pattern of division. We have investigated the establishment of these axes by following the movement of the centrosomes. Centrosome separation follows a reproducible pattern in all cells, and this pattern by itself results in an orthogonal pattern of cleavage. In those cells that divide on the same axis, there is an additional directed rotation of pairs of centrosomes together with the nucleus through well-defined angles. Intact microtubules are required for rotation; rotation is prevented by inhibitors of polymerization and depolymerization of microtubules. We have examined the distribution of microtubules in fixed embryos during rotation. From these and other data we infer that microtubules running from the centrosome to the cortex have a central role in aligning the centrosome-nuclear complex.     

Embryonic development of insect eggs formed without follicular epithelium

In the paedogenetic Dipteran insect Heteropeza pygmaea it is possible by physical or chemical means to obtain oocyte-nurse chamber complexes lacking the follicular epithelium. Such oocytes nevertheless complete oogenesis and begin embryonic development. Development of these “naked” eggs has been compared to normal egg development by cinematographic analysis. Eggs which are formed without follicular epithelium are completely spherical in shape and the increase in size which normally occurs during cleavage is much less extensive. Naked eggs show shape changes during the first part of cleavage, in which bulgy cytoplasmic protrusions are formed and disappear continuously. Protrusions which are present during the mitotic divisions are partly cleaved. Cleavage folds occur much earlier in naked eggs than in normal eggs. On the other hand, the duration of the mitotic cycles during nuclear multiplication of normal and naked eggs is similar. Development of naked eggs usually continues for some time after blastoderm formation before degeneration sets in. The events taking place prior to embryonic death are difficult to relate to normal gastrulation events. However, in some cases the morphogenetic movements of naked embryos resemble germ band formation of normal embryos.     

Subcortical microtubule network separates the periplasm from the endoplasm and is responsible for maintaining the position of accessory nuclei in hymenopteran oocytes

Oocytes of hymenopterans are equipped with peculiar organelles termed accessory nuclei. These organelles originate from the germinal vesicle (oocyte nucleus) and gather preferentially at the anterior pole. To gain insight into the mechanism of uneven (asymmetrical) distribution of accessory nuclei, the organization of the microtubule cytoskeleton in the oocytes of two hymenopterans Chrysis ignita and Cosmoconus meridionator has been studied. It is shown that during late previtellogenesis two networks of microtubules are present along the contact zone between the oocyte and enveloping follicular epithelium. The external one is associated with belt desmosomes connecting neighbouring follicular cells. The internal network is composed of randomly orientated microtubules and separates transparent, organelle-free periplasm from the endoplasm. All cellular organelles and the germinal vesicle are localized in the endoplasm. Accessory nuclei are accumulated in the anterior endoplasm; they always lie in direct contact with the subcortical network. Treatment with colchicine results in the disappearance of the periplasm as well as in the redistribution of cellular organelles including accessory nuclei. Presented findings suggest that subcortical microtubules play an important role in the positioning of accessory nuclei throughout the ooplasm.     

Monoplastidic mitosis in the mossTimmiella barbuloides (Bryophyta)

Summary Mitotic cell division of monoplastidic sporogones was investigated in the mossTimmiella barbuloides (Brid.) Moenk. (Pottiales, Bryophyta) by TEM. Division polarity of sporogones is established by the interphase position of the single oblong cup-shaped plastid, which is orientated with its long axis parallel to one of the cell walls. In preprophase the plastid elongates and its extremities bend at right angles. Plastid growth is directed by microtubules and accompanied by plastid tubules. The plastid begins the process of duplication by constricting centrally in the plane of the future cytokinetic septum. There is no preprophase band of microtubules at the division site. The large central nucleus becomes fusiform and aligned parallel to the main plastid axis. By the end of prophase the daughter plastids are positioned at the opposite poles of the nucleus where they probably function as nucleating or organizing centres for the spindle microtubules. Metaphase and anaphase spindles contain long sheets of ER. Cytokinesis involves the formation of a well developed phragmoplast.Abbreviations TEM transmission electron microscopy - PPB preprophase band of microtubules - ER endoplasmic reticulum     

Structure of the ovaries and follicular epithelium morphogenesis in Drosophila and its kin

In insects, the ovarian follicular epithelium morphogenesis has been intensively studied and best characterized in the fruit fly, Drosophila melanogaster. It is well established that initially identical somatic follicular cells (FCs) form a simple epithelium overlying the germline cells, but during oogenesis, they diversify into a number of morphologically distinct subpopulations each responsible for creating specific eggshell structures. In addition, some FC subpopulations (e.g. polar cells) are indispensable in establishing antero-posterior and dorso-ventral ovarian follicle axes and patterning of the developing embryo. The morphological and molecular changes that occur during follicular epithelium morphogenesis in Drosophila are frequently considered as a paradigm of the FC diversification in all flies. However, recent comparative studies indicate that, in dipterans, the functioning of the ovarian follicles is diverse, group-specific and may significantly differ from the Drosophila model system. We discuss the similarities and differences of the ovary structure and follicular epithelium morphogenesis in different dipteran groups and put them into a phylognetic context. We suggest that the migratory activity of the FCs represents an evolutionary novelty that evolved in the ancestors of higher dipterans (Brachycera). Subsequently, during evolution of this subgroup, the number of migrating FC subpopulations has gradually increased from one (in Orthorrhapha) to four (in Cyclorrhapha).     

Division polarity,development and configuration of microtubule arrays in Bryophyte meiosis

Summary Immunofluorescence and TEM studies of meiosis in two mosses (Bryophyta) provide evidence that the prophasic tetrahedral system of microtubules contributes directly to the metaphase I spindle. Intense staining of tubulin, conspicuously absent around the nuclear envelope, is first seen associated with plastids. By mid-prophase, microtubules radiate from the plastids to the nuclear envelope and become organized into six bands that interconnect the four plastids, forming a tetrahedral cytoskeleton surrounding the nucleus. During transition of prophase to metaphase, the four poles of the tetrahedral microtubule system converge in pairs toward opposite cleavage furrows. Opposite furrows occupy mutually perpendicular planes and the pair of microtubule focal points straddling one furrow lies at right angles to the pair straddling the opposite furrow. Additional microtubules terminate in numerous small clusters in the concave polar regions arching over the cleavage furrows. By early anaphase, the microtubule focal points lie very close to the division axis. We conclude that microtubules recruited from the prophasic quadripolar system are incorporated into the mature metaphase I spindle and the two principal focal points at each pole are those derived from poles of the prophasic quadripolar system.     

Aster self-organization at meiosis: a conserved mechanism in insect parthenogenesis?

Unfertilized eggs usually lack maternal centrosomes and cannot develop without sperm contribution. However, several insect species lay eggs that develop to adulthood as unfertilized in the absence of a preexisting centrosome. We report that the oocyte of the parthenogenetic viviparous pea aphid Acyrthosiphon pisum is able to self-organize microtubule-based asters, which in turn interact with the female chromatin to form the first mitotic spindle. This mode of reproduction provides a good system to investigate how the oocyte can assemble new centrosomes and how their number can be exactly monitored. We propose that the cooperative interaction of motor proteins and randomly nucleated surface microtubules could lead to the formation of aster-like structures in the absence of pre-existing centrosomes. Recruitment of material along the microtubules might contribute to the accumulation of pericentriolar material and centriole precursors at the focus of the asters, thus leading to the formation of true centrosomes. The appearance of microtubule asters at the surface of activated oocytes could represent a possible common mechanism for centrosome formation during insect parthenogenesis.     

Polarization of eggs and embryos: some common principles

Embryonic development depends on the establishment of polarities which define the axial characteristics of the body. In a small number of cases such as the embryo of the fly drosophila, developmental axes are established well before fertilization while in other organisms such as the nematode worm C. elegans these axes are set up only after fertilization. In most organisms the egg posesses a primary (A-V, Animal-Vegetal) axis acquired during oogenesis which participates in the establishment of the embryonic axes. Such is the case for the eggs of ascidians or the frog Xenopus whose AV axes are remodelled by sperm entry to yield the embryonic axes. Embryos of different species thus acquire an anterior end and a posterior end (Antero-Posterior, A-P axis), dorsal and ventral sides (D-V axis) and then a left and a right side.     

鹤顶兰胚囊发育过程中微管变化的共焦显微镜观察

光镜的观察确定了鹤顶兰（Ｐｈａｉｕｓ ｔａｎｋｅｒｖｉｌｌｉａｅ （Ａｉｔｏｎ） Ｂｌ．）胚囊发育属单孢子蓼型。应用免疫荧光标记技术及共焦镜观察了胚囊发育过程中微管分布的变化。当孢原细胞初形成时,细胞内的微管呈网状分布。之后,孢原细胞体积增大发育为大孢子母细胞。大孢子母细胞延长,进入减数分裂Ⅰ。微管由分裂前的网状分布变为辐射状排列。二分体的两个细胞内的微管分布一样,呈辐射状。四分体的近珠孔端的３ 个大孢子解体,细胞内的微管消失。靠合点端的功能大孢子内有许多微管呈网状分布。当功能大孢子进入第一次有丝分裂时,细胞内的微管由网状变为辐射状,从核膜伸展至周质。再经两次有丝分裂形成八核胚囊。在核分裂之前微管一般是呈网状分布并紧包围着核。在分裂期间二核和四核胚囊都呈极性现象,微管系统也呈极性分布。微管在八核胚囊内的分布变化情形特别复杂。首先,八核分别作不同程度的移动,其中两个核移向胚囊中央,珠孔端和合点端的３ 个核分别互相靠拢,形成３ 个区,即中央区、反足区和卵器区。胚囊未形成区时,８ 个核都被网状分布的微管包围着。当胚囊明显分成区时,反足区内的微管仍作网状分布。中央区的微管分布则趋疏松,形成篮形结构,包围着液泡和两个极核。在     

Confocal Microscopic Observations on Microtubular Cytoskeleton Changes During Megasporogenesis and Megagametogenesis in Phaius tankervilliae (Aiton) Bl.

In nun orchid (Phaius tankervilliae (Alton) B1. ) embryo sac development follows the monosporic pattern. Changes in the pattern of organization of the microtubular cytoskeleton during megasporogenesis and megagametogenesis in this orchid were studied using the immunofluorescence technique and eonfocal microscopy. At the initial stage of development the microtubules in the arehesporium were randomly oriented into a network. Later the archesporial cell elongated to form the megasporocyte. The cytoskeleton in the elongated megasporoeyte was radially organized in which microtubules extending from the nuclear envelope to the peripheral region of the cell. The megasporoeyte then underwent meiosis 1 to form a dyad. The dyad cell at the chalazal end was larger than the cell at the micropylar end. Microtubules in the dyad cell were radially oriented. The dyad underwent meiosis to give rise to a linear array of four megaspores (i. e. tetrad formation). The chalazal-far most megaspore survived and became the functional megaspore, which contained a set of randomly oriented microtubules. The microtubules in the other 3 megaspore disappeared as the cells degenerated. The functional megaspore then underwent mitotic division giveing rise to a 2 nucleate embryo sac. The nuclei of the 2-nucleate embryo sac were separated by a set of longitudinally oriented microtubules which ran parallel to the long axis of the embryo sac. Each nucleus in the embryo sac was surrounded by a set of perinuelear microtubules. The gnucleate embryo sac again underwent mitotic division to form a 4-nucleate embryo sac. The division of the two nuclei was synchronous. But the orientation of the division plan of the two spindles was different (i. e. the spindle microtubules at the chalazal end ran parallel with the long axis of the embryo sac and those at the mieropylar end ran at right angle to the axis of the embryo sac). The 4 nuclei of the 4-nucleate embryo sac were all tightly surrounded by randomly oriented microtubules. Later the paired nuclei at the micropylr end and at the chalazal end as well underwent mitotic division in seguence. At this time when the embryo sac had reached the 8-nucleate embryo sac stage. The pattern of organization of the microtubules was very complex. Initially the nuclei were surrounded by a set of randomly oriented microtubules, but after the two polar nuclei had moved to the central region of the embryo sac, three different organizational zones of microtubules appeared, viz: a randomly oriented set of microtubules surrounding each nucleus in the chalazal zone: a set (in the form of a basket) of cortical microtubules which surrounded the vacuoles and the two polar nuclei in the central zone and a loosely knitted network of microtubules surrounding the nucleus that later became the egg cell nucleus in the micropylar zone. The two nuclei that would become the nuclei of the synergids were surrounded by a set of more densely packed mierotubules. Towards far the most micropylar end some microtubules formed thick bundles. The site of appearance of these thick bundles coincided with the site of development of the filiform apparatus. The pattern of microtubule organization after cellularization (i. e. at the beginning of embryo sac maturation) did not change much. The author's results indicated that various patterns of microtubule organization observed in the developing embryo sac of nun orchid reflected the complexity and dynamism of the embryo sac.     

Confocal scanning laser microscopy of the follicular epithelium in ovarioles of the stick insect Carausius morosus

Ovarian follicles of the stick insect Carausius morosus were analyzed by confocal laser microscopy and immunocytochemistry with a view to studying cell polarity in the follicular epithelium. Such probes as anti-α-tubulin antibodies and Rh-phalloidin were employed to establish how the follicle cell cytoskeleton changes during ovarian development. Data show that α-tubulin prevails over the basal end, while F-actin appears more abundant along the apical end of the follicle cells. This finding was further corroborated by immunogold cytochemistry, showing that label along the basal end is primarily associated with microtubules, while that along the apical end is due to follicle cell microvilli interdigitating with the oocyte plasma membrane. A monoclonal antibody specifically raised against a vitellin polypeptide was used to investigate the role the follicular epithelium might play in relation to vitellogenin (Vg) uptake by the oocyte. Data show that under these conditions label is restricted to the intercellular channels of the follicular epithelium, thus providing further support to the notion that Vg enters the oocyte through the extracellular pathway leading from the basement lamina to the oocyte surface. By contrast, the use of a monoclonal antibody raised against a fat-body-derived protein of 85 kDa that is specifically sulfated within the follicle cells provides evidence for the existence of an alternative way of gaining access to the oocyte surface, that is by transcytosis through the follicular cell epithelium. These findings confirm our earlier observations on stick insect ovarioles whereby polarization in the follicular epithelium is primarily addressed to sustain a transcytotic vesicular traffic between opposite poles of the follicle cell of Vg toward the oocyte surface.     

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.     

Theoretical evidence that more microtubules reach the cortex at the pole than at the equator during anaphase in sea urchin eggs

Astral microtubules are rapidly elongated during anaphase and telophase in sea urchin eggs. The number of microtubules extending to the cell surface was calculated with a computer. For the calculations, microtubules were assumed to radiate from the astral center uniformly over angles. Although microtubules from two asters freely overlapped around the equator, the number per the unit area, i.e. the surface density, was larger in the polar region than in the equatorial region. The ratio of the theoretically calculated numbers in the two regions was close to the ratio obtained from the ultrastructural observations by Asnes and Schroeder in 1979. When counted in the longitudinal section including two astral centers, the microtubule number was a little larger in the equatorial region than in the polar region. However, the numbers do not represent the surface density because the two-dimensional section contains only a small portion of all the microtubules spreading in the three-dimensional space. The fluorescence image for tubulin, in most cases, provides the microtubule distribution in the longitudinal section. Therefore, from such an image, we cannot judge whether the surface density of astral microtubules is larger at the pole or at the equator.     

All meiotic chromosomes and both centrosomes at spindle pole in the zygotes discarded as two polar bodies in clam Corbicula leana: unusual polar body formation observed by antitubulin immunofluorescence

To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against α-tubulin, γ-tubulin, and 4’-6’-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two ”first polar bodies” and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster. Received: 29 September 1999 / Accepted: 24 November 1999