Structural and functional polarity of starfish blastomeres

The cortex of the blastomeres of Asterina pectinifera are structurally polarized so that some kinds of granules in the cortex, which can be stained vitally with Nile blue (Nile blue-positive granules, NBGs), and microvilli were distributed mainly in the apical region. The blastomeres always faced the adjoining blastomeres and blastocoel with the NBG-free, smooth region during embryogenesis. To confirm whether such blastomeres are functionally polarized, we rotated one of the blastomeres in the 2-cell-stage embryo so that it faced the other with the NBG-containing region. As a result, all embryos developed into twin or partitioned blastulae. This shows that the blastomeres are functionally polarized and have to orient the basal cortex toward the inner side of the embryo in order to be integrated into a blastula together with the others. The cortical polarity was formed and maintained even in blastomeres of dissociated embryos. In such blastomeres the cleavage furrows were formed along the axis of polarity. When the blastomeres began to adhere closely to each other at the 256-cell stage, only the NBG-free (basal) region acquired adhesiveness. These facts make it possible to infer why the correct apicobasal orientation of blastomeres is necessary for embryonic integration, without considering intercellular communication during the cleavage stage.     

Induction of cytoplasmic polarity in heterokaryons of mouse 4-cell-stage blastomeres fused with 8-cell- and 16-cell-stage blastomeres

Cell surface and cytoplasmic polarity is exhibited by the blastomeres of mouse preimplantation embryos following compaction at the 8-cell stage of cleavage. It has been hypothesized that cytoplasmic polarity is initiated by plasma membrane functions of polar blastomeres that are absent from apolar blastomeres. To test this hypothesis the plasma membranes of "test" polar and apolar 8-cell- and 16-cell-stage blastomeres were inserted into the plasma membrane of "carrier" 4-cell-stage blastomeres by polyethylene glycol-mediated fusion of carrier-test blastomere pairs. After a 4-hr culture period each heterokaryon was scored for the distribution of two marker organelles--lipid droplets and nuclei--with respect to their proximity to the plasma membrane insert from the test blastomere. Plasma membrane inserts from polar test blastomeres were identified by labeling their apical domains with fluorescently tagged (succinylated) concanavalin A. The incidence of polar heterokaryons (those exhibiting a discrete fluorescently labeled area of plasma membrane corresponding to the apical domain inherited from the test blastomere) was 55/85 (69%) and 48/79 (61%) for 8-cell-stage and 16-cell-stage test blastomeres, respectively. In all polar heterokaryons, both nuclei were subjacent to the fluorescent label (apical domain of a polar plasma membrane insert), while the majority of lipid droplets resided in the hemisphere opposite the fluorescent label. In all 61 apolar heterokaryons examined (those lacking a discrete fluorescently labeled plasma membrane area) both nuclei were centrally located and lipid droplets were randomly distributed. These observations are consistent with the hypothesis that cytoplasmic polarity can be initiated by properties that distinguish the plasma membranes of polar blastomeres from those of apolar blastomeres.     

Redistribution of microtubules and pericentriolar material during the development of polarity in mouse blastomeres

The distribution of microtubules and microtubule organizing centers (MTOCs) during the development of cell polarity in eight-cell mouse blastomeres was studied by immunofluorescence and immunoelectron microscopy using monoclonal anti-tubulin antibodies and an anti-pericentriolar material (PCM) serum. In early eight-cell blastomeres microtubules were found mainly around the nucleus and in the cell cortex, whereas PCM foci were observed dispersed in the cytoplasm. During the eight-cell stage, microtubules disappeared from the area adjacent to the zone of intercellular contact and accumulated in the apical part of the cell while their number decreased in the basal domain. The PCM also relocalized to the apical domain of the cell, but this occurred after the redistribution of the microtubules by a mechanism that involved the microtubule network. The possible roles of both MTOCs and microtubules in establishing cell polarity are discussed.     

Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo

Membrane topography and organization of cortical cytoskeletal elements and organelles during early embryogenesis of the mouse have been studied by transmission and scanning electron microscopy with improved cellular preservation. At the four- and early eight-cell stages, blastomeres are round, and scanning electron microscopy shows a uniform distribution of microvilli over the cell surface. At the onset of morphogenesis, a reorganization of the blastomere surface is observed in which microvilli becomes restricted to an apical region and the basal zone of intercellular contact. As the blastomeres spread on each other during compaction, many microvilli remain in the basal region of imminent cell-cell contacts, but few are present where the cells have completed spreading on each other. Microvilli on the surface of these embryos contain linear arrays of microfilaments with lateral cross bridges. Microtubules and mitochondria become localized beneath the apposed cell membranes during compaction. Arrays of cortical microtubules are aligned parallel to regions of apposed membranes. During cytokinesis, microtubules become redistributed in the region of the mitotic spindle, and fewer microvilli are present on most of the cell surface. The cell surface and cortical changes initiated during compaction are the first manifestations of cell polarity in embryogenesis. These and previous findings are interpreted as evidence that cell surface changes associated with trophoblast development appear as early as the eight-cell stage. Our observations suggest that morphogenesis involves the activation of a developmental program which coordinately controls cortical cytoplasmic and cell surface organization.     

Contact-independent polarization of the cell surface and cortex of free sea urchin blastomeres

In a normal, intact sea urchin embryo blastomeres are structurally polarized so that all microvilli and cortical "pigment granules" are situated at the apical surfaces facing the hyaline layer and are absent from basolateral surfaces facing adjacent blastomeres and the internal embryonic cavity. To test the roles of intercellular contacts and the hyaline layer in the process of establishing this blastomere polarity, these two factors were experimentally eliminated; sea urchin eggs of four species were denuded of the nascent hyaline layer soon after fertilization and then cultured in calcium-free artificial seawater to prevent subsequent intercellular adhesion and contact. Such free blastomeres divided normally and still developed polarized distributions of microvilli and pigment granules resembling those of the corresponding blastomeres in intact embryos. These results indicate that the process of polarization is intrinsic to individual blastomeres (self-polarization) and that neither intercellular contacts nor adhesion of microvilli to the hyaline layer is necessary. The precise temporal and spatial coincidence of the patterns of polarization and the division cycles further suggests that a mechanistic link is maintained among cell division, blastomere polarization, and probably also a heritable component of the animal-vegetal axis.     

Localization of an Exogastrula-Inducing Peptide (EGIP) in Embryos of the Sea Urchin Anthocidaris crassispina

Exogastrula-inducing peptides (EGIPs) are present in the unfertilized eggs and embryos of the sea urchin Anthocidaris crassispina . They induce exogastrulation when added exogenously to the embryos. The localization of EGIP-D during embryogenesis has been explored using polyclonal antibodies against EGIP-D. Immunofluorescent staining revealed that EGIP-D is stored in the cytoplasm of immature oocytes and is concentrated into vesicles in unfertilized eggs. At fertilization, the vesicles containing EGIP-D (EGIP-vesicles) migrate to the cortical surface of the zygotes and are distributed in a ring-like pattern at the apical surface of blastomeres, disappearing from basal surfaces and those adjacent to neighboring cells, during development from cleavage stages to larval stages. Mesenchyme cells also contain the vesicles but no such polarized distribution of vesicles is apparent. Acidic vesicles with a similar polarized distribution were examined by staining with acridine orange, which revealed that acidic vesicles were in close proximity to the surface of eggs at fertilization and were then distributed in a ring-like pattern at the apical surface of blastomeres as are the EGIP-vesicles. Furthermore, immunoelectron microscopy revealed that EGIP-D is present in vesicles that are located at the apical surface of blastomeres. The significance of the localized distribution of EGIP-D is discussed in relation to its function.     

Expression of murine early embryonic antigens, SSEA-1 and antigenic determinant of EMA-1, in embryos and ovarian follicles of a teleost medaka (Oryzias latipes)

Stage-specific embryonic antigen-1 (SSEA-1) and the antigenic determinant of monoclonal antibody EMA-1 are expressed in a stage-specific manner in mouse early embryos. To study whether these antigens generally exist in fish, expression of the antigens was examined in embryos, ovarian follicles, and adult tissues of a teleost medaka (Oryzias latipes), using immunohistochemical techniques. In 1-cell-stage embryos, these carbohydrate antigens were found in numerous cytoplasmic granules in the blastodisc and the cortical cytoplasm. These granules gradually decreased in number as the embryos developed. In 4-cell-stage embryos, the antigens appeared on the cleavage planes and were located on the cleavage planes within the blastoderm in the following cleavage stages. In blastula-stage embryos, the expression was ubiquitously found on the cell surface of blastomeres. At the mid-gastrula stage, the antigens were restricted to the enveloping layer, yolk syncytial layer, and cortical cytoplasm, but were rarely found in deep cells that contribute to formation of the embryonic body. In later-stage embryos and adult fish, the antigens were located in various tissues. In ovarian follicles, the antigens were found in granules of oocytes and granulosa cells. These observations were basically consistent with those in mice; however, expression in 1-cell-stage embryos and ovarian follicles has not been observed in mice. This unexpected finding suggests that the antigens are produced in granulosa cells and transferred to 1-cell-stage embryos via oocytes, and that the antigens involved in the early developmental process are maternally prepared in teleosts.     

The organization of actin filaments in human placental villi

The surface of the syncytial trophoblast of the human placenta is covered by a microvillous (brush) border that is in direct contact with maternal blood. Because of this location, it is the site of a variety of transport, enzymatic and receptor activities vital to many placental functions. The organization of the brush border as well as other features of placental villus organization may well be influenced by the distribution of cytoplasmic actin filaments. In order to determine the distribution of actin filaments in human placenta, small pieces of villi were briefly fixed in glutaraldehyde, permeabilized with saponin, and incubated in solutions containing subfragment 1 of myosin (S1). After S1 decoration of actin filaments, tissue was fixed in glutaraldehyde containing tannic acid in order to better visualize the polarity of the filaments, and prepared for electron microscopic examination. The microvilli each contained a core of actin filaments running from the tip of the microvillus to the apical cytoplasm. Most of the actin filaments displayed a distinct polarity, with the S1 arrowheads pointing away from the microvillar tips. These filaments extended only a short distance into the apical cytoplasm. There appeared to be another group of actin filaments in a matlike arrangement in the apical cytoplasm. Coated pits and vesicles were often observed between the microvilli. There appeared to be no clear association between the coated pits and decorated actin filaments, but this was difficult to establish with certainty because of the close proximity of the microvilli. Bundles of actin filaments were sometimes observed near the basal cell surface of the syncytial trophoblast, and in pericytes and capillary endothelial cells in the cores of the villi.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytoskeleton, endocytosis and cell polarity in the mouse preimplantation embryo

The process of cell polarization in mouse 8-cell embryos includes the formation of a polar cluster of cytoplasmic endocytotic organelles (endosomes) subjacent to an apical surface pole of microvilli. A similar polar morphology, supplemented by basally localized secondary lysosomes, is evident following division to the 16-cell stage in outside blastomeres, precursors of the trophectodermal lineage. The roles of microfilaments and microtubules in generating and stabilizing endocytotic and surface features of polarity (visualized by horseradish peroxidase incubation and indirect immunofluorescence labeling, respectively) have been evaluated by exposure of 8- and 16-cell embryos and 8-cell couplets to drugs (cytochalasin D, colcemid, nocodazole) that disrupt the cytoskeleton. The generation of endocytotic polarity is dependent upon intact microtubules and microfilaments, but the newly established endocytotic pole in blastomeres from compacted 8-cell embryos appears to be stabilized exclusively by microtubules. Polarized endocytotic organelles at the 16-cell stage are more resistant to drug treatment than at the 8-cell stage (probably due to microfilament interactions) indicating a maturation phase in the polar cell lineage. Microtubules are also responsible for the orientation of endocytotic clusters along the cell's axis of polarity. In contrast, the generation and stability of polarity at the cell surface appears relatively independent of cytoskeletal integrity. The results are discussed in relation to the mechanisms that may control the development and stabilization of polarization during cleavage.     

Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres.

Mouse preimplantation embryos were examined for the cell surface expression of epitopes that cross-react with antibodies to a 75-kDa subunit of a purified porcine renal brush border Na+/glucose cotransport system. A Na+ cotransport system is hypothesized to reside in the apical plasma membrane domain of mouse polar blastomeres and to be associated with the induction of their apical-basal polarity. Western blot analysis showed that unfertilized oocytes as well as preimplantation embryos contain a cross-reacting antigen with an apparent molecular weight of about 75,000. Embryos and their isolated blastomeres were double-labeled and assayed by indirect immunofluorescence (IIF) for the expression of epitopes (visualized by labeling with rabbit antiserum or mouse monoclonal IgG to cotransporter followed by the appropriate rhodamine-conjugated second antibodies) and for the development of cell surface polarity (visualized by the apical restriction of fluoresceinated succinylated concanavalin A binding; FS Con A). IIF did not detect these epitopes until after the second cleavage when 4-cell embryos expressed low-to-moderate levels. Although epitopes were expressed on all surfaces of 4-cell blastomeres, some blastomeres expressed more epitopes on their apical surfaces than on their basolateral ones. All precompaction 8-cell embryos expressed epitopes, with expression being greater apically on some blastomeres. The level of expression appeared to reach a maximum on morulae and to decline on cavitating embryos. Assays performed on isolated blastomeres from postcompaction embryos showed that by the 16-cell stage epitope expression appeared to become restricted to FS Con A-labeled apical plasma membrane domains and was no longer evident on basolateral domains. This apparent apical restriction of epitope expression was confirmed by electron microscopic examination of immunogold-labeled isolated polar 16-cell blastomeres. These results demonstrate that preimplantation mouse embryos contain an antigen(s) that is immunologically and structurally similar to a 75-kDa renal Na+/glucose cotransporter. The onset of cell surface expression of this antigen precedes development of the stable polar phenotype.     

Scanning microscopy of disaggregated and aggregated preimplantation mouse embryos

Summary Blastomeres isolated from 8-and 16-cell embryos (that is 1/8 and 1/16) show a smooth surface at their point of contact with other blastomeres and a microvillous free surface. Microvilli reappear completely on the smooth surface of 52% of 1/8 embryos and partially on 88% of 1/16 embryos if cultured in vitro for 6 h. When 2-to 8-cell embryos are aggregated to 8-cell embryos and forced apart after 1–3 h, the contact surface of the 8-cell embryos has become smooth. Fixed 8-cell embryos are also able to induce complete disappearance of microvilli on the contact surface of a living 8-cell embryo. Embryos having more than 8 cells do not induce complete disappearance of microvilli on the contact surface of 8-cell embryos. Aggregates of late morulae do not show complete disappearance of microvilli at their contact surfaces but rather a loosening of their peripheral blastomeres.Our results show that isolated 1/8 and 1/16 embryos tend to recover from regionalization, that the process of aggregation of embryos having 8 cells or less is similar to compaction and that embryos having more than 8 cells seem to aggregate by cell sorting. The processes of compaction, adhesion and reassortment are briefly discussed. We submit that blastomere regionalization, which depends on cell to cell contact, may be the spatial basis of embryonic regulation and of the inside-outside normal differentiation of early mouse embryos.     

Carbohydrate cytochemistry of the intestinal epithelium of Ascaris suum. Nature of the microvilli glycocalyx and basal lamella.

Results of various cytochemical tests demonstrate large deposits of glycogen within the intestinal absorptive cells of Ascaris suum. Carbohydrate material is also associated with the microvilli surface and basal lamella. Staining produced by the periodate-thiocarbohydrazide-osmium procedure was abolished by analine or m-aminophenol. Diastase digestion did not alter the staining on the microvilli surface. Similar results were seen using the silver methenamine procedure. A positive reaction was noted on the microvilli surface, vesicles in both the apical and basal cytoplasm, Golgi apparatus, and basal lamella. Lanthanum nitrate stained the microvilli surface and intercellular spaces between absorptive cells. Alcian blue or cetylpyridinium chloride in combination with lanthanum enhanced the staining produced by lanthanum alone. These results suggest the presence of acidic glycans on both the microvilli surface and basal lamella.     

Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development

Actin is one of the major proteins in mammalian oocytes. Most developmental events are dependent on the normal distribution of filamentous (F-) actin. Polymerization of nonfilamentous (G-) actin into F-actin is important for both meiosis and mitosis. This study examined G- and F-actin distribution in pig oocytes and embryos by immunocytochemical staining and confocal microscopy. Actin protein was quantified by electrophoresis and immunoblotting. G-Actin was distributed in the whole cytoplasm of oocytes and embryos irrespective of their stages. F-Actin was distributed at the cortex of oocytes and embryos at all stages, at the joint of blastomeres in the embryos, in the cytoplasm around the germinal vesicle (GV), and in the perinuclear area of 2- to 4-cell-stage embryos. No differences in the amount of actin protein were found among oocytes and embryos. Oocytes cultured in medium with cytochalasin D (CD), an inhibitor of microfilament polymerization, underwent GV breakdown and reached metaphase I but did not proceed to metaphase II. Two- to 4-cell-stage embryos cultured in medium with CD did not develop to blastocysts. When GV-stage oocytes or 2- to 4-cell-stage embryos treated with CD for 6 h were re-cultured in media without CD, oocytes or embryos re-assembled actin filaments and underwent a meiotic maturation or blastocyst formation similar to that of controls. These results indicate that it is the polymerization of G-actin into F-actin, not actin protein synthesis, that is important for both meiosis and mitosis in pig oocytes and embryos.     

Cell polarity in sea urchin embryos: reorientation of cells occurs quickly in aggregates

Four apical components were used as markers for the apical end of the cell in studies centering on cell polarity in the early blastula stage of sea urchin embryos and in aggregates of cleavage stage cells. Cells were observed to maintain their polarity for several hours if dissociated and cultured in suspension. Orientation of cells in aggregates initially is random; however, within 3 hr the cells have reoriented so that their apical-basal axis corresponds to the correct inside-outside position in the aggregate. This reorientation occurs before formation of a basal lamina or a new hyalin layer in the aggregate, and appears to take place by a rotation or other movement of individual cells. The polarity within each cell is maintained during reorientation. An apical surface antigen is colocalized with concentrations of filamentous actin. Treatment of isolated cells with cytochalasin B causes the antigen to lose its apical position and eventually become distributed around the outside of the cell. Microtubules are visible radiating from two foci closely associated with the nucleus in untreated cells. Treatment of isolated cells with nocodazole leaves the apical cell surface marker and its associated actin undisturbed, but causes the nucleus to lose its apical position. Cytochalasin B and colchicine both prevent reorientation of cells in aggregates. Thus polarity appears to be a constant for the cells, and their reorientation in aggregates occurs prior to the polarized release of extraembryonic matrix and basal lamina.     

Periodic appearance and disappearance of microvilli associated with cleavage cycles in the egg of the ascidian, Halocynthia roretzi

The surface of eggs of the ascidian Halocynthia roretzi, observed with SEM, is essentially smooth until immediately before cell division when numerous microvilli appear and remain during cytokinesis. As the dividing blastomeres become closely adherent, however, the microvilli disappear and the eggs recover their smooth surface. This periodic appearance-disappearance of microvilli is repeated at each cleavage cycle up to at least the 32-cell stage. During blastomere adhesion, microvilli that have appeared near the plane of the first cleavage or of the bilateral symmetry seem to fuse together across the plane to form a zipper-like complex of cytoplasmic processes, which might be responsible for attachment of the two halves of these bilaterally symmetrical embryos via the blastomeres bordering the plane of symmetry.     

Ultrastructure of the Male Accessory Glands and Sperm Ducts of Cylindrotaenia hickmani(Cestoda,Cyclophyllidea)

Abstract The ultrastructure of unicellular accessory glands (= prostate glands) and external male ducts of the cestode Cylindrotaenia hickmaniare described. Accessory glands open into the lumen of the external common sperm duct (= external vas deferens). The gland cells contain abundant endoplasmic reticulum, Golgi bodies and secretory bodies, and have elongate necks that pierce the apical cytoplasm of the duct. Cell contact with the apical cytoplasm of the sperm duct is mediated by septate desmosomes. Accessory glands secrete spherical particles, with a diameter of approximately 70 nm, that adhere to spermatozoa. The roles of these accessory glands may relate to activity of the sperm or development of the female system after insemination. Paired sperm ducts arise from testes, and unite to form a common sperm duct. Each duct consists of a tubular anucleate cytoplasmic region which is supported by nucleated cytons that lie sunken in the parenchyma. The apical cytoplasm of the paired sperm ducts (= vasa efferentia) possesses apical microvilli and abundant mitochondria, but few other cytoplasmic features. The apical cytoplasm of the common sperm duct possesses sparse apical microvilli and numerous electronlucent vesicles. The male gonoducts form an elongate syncytium which is markedly polarized along the length of the ducts. The ducts also display apical–basal polarity in that sunken nucleated cytons support the apical cytoplasm which in turn has distinct basal and apical domains.     

Development of rat tetraploid and chimeric embryos aggregated with diploid cells

In the present study, we examined the preimplantation and postimplantation development of rat tetraploid embryos produced by electrofusion of 2-cell-stage embryos. Developmental rate of tetraploid embryos to morula or blastocyst stage was 93% (56/60) and similar to that found in diploid embryos (95%, 55/58). After embryo transfer, rat tetraploid embryos showed implantation and survived until day 8 of pregnancy, however the conceptuses were aberrant on day 9. In mouse, tetraploid embryos have the ability to support the development of blastomeres that cannot develop independently. As shown in the present study, a pair of diploid blastomeres from the rat 8-cell-stage embryo degenerated immediately after implantation. Therefore, we examined whether rat tetraploid embryos have the ability to support the development of 2/8 blastomeres. We produced chimeric rat embryos in which a pair of diploid blastomeres from an 8-cell-stage green fluorescent protein negative (GFP-) embryo was aggregated with three tetraploid blastomeres from 4-cell GFP-positive (GFP+) embryos. The developmental rate of rat 2n(GFP-) <--> 4n(GFP+) embryos to the morula or blastocyst stages was 93% (109/117) and was similar to that found for 2n(GFP-) <--> 2n(GFP+) embryos (100%, 51/51). After embryo transfer, 2n(GFP-) <--> 4n(GFP+) conceptuses were examined on day 14 of pregnancy, the developmental rate to fetus was quite low (4%, 4/109) and they were all aberrant and smaller than 2n(GFP-) <--> 2n(GFP+) conceptuses, whereas immunohistochemical analysis showed no staining for GFP in fetuses. Our results suggest that rat tetraploid embryos are able to prolong the development of diploid blastomeres that cannot develop independently, although postimplantation development was incomplete.     

Organization of actin microfilaments in the apical border of oviduct ciliated cells

Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the presence of numerous cilia and microvilli at their apical pole. Most S1-decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens. Together with the other cytoskeletal elements, the microfilaments increase ciliary anchorage and could be involved in the coordination of ciliary beating. Moreover, microvilli surrounding the cilia probably modify ciliary beating by offering resistance to cilium bending. The presence of microvilli could explain the fact that mainly the upper part of the cilia appanars to be involved in the axonemal bending in metazoan ciliated cells.     

Effects of phlorizin and ouabain on the polarity of mouse 4-cell/16-cell stage blastomere heterokaryons

Cell polarity is thought to be required for the efficient production of nascent blastocoele fluid, which begins at the 16-cell stage of mouse preimplantation development. In this study the 4-cell/16-cell blastomere heterokaryon was used to test the hypothesis that solute transport across the apical membrane domain induces the apical-basal axis of organelle distribution across polar 16-cell-stage blastomeres. Fusion of 4-cell/16-cell blastomere pairs resulted in a population of heterokaryons of which 65% were polar (contain an apical plasma membrane domain from a polar 16-cell-stage plasma membrane insert) and 30% were apolar (contain an apolar 16-cell-stage plasma membrane insert). Polar heterokaryons were distinguished from apolar ones by labeling their apical domains with fluorescent succinylated concanavalin A. In polar heterokaryons, both nuclei (labeled with Hoeschst 33242) were immediately subjacent to the apical plasma membrane domain, while in apolar heterokaryons both nuclei were located centrally. Two inhibitors of apical transmembrane solute transport--phlorizin, which inhibits brush border (apical) Na+/glucose symporters, and ouabain, which inhibits Na+/K+-ATPase, thereby modifying the transmembrane Na+ gradient--were examined for their effect on nuclear position in polar and apolar heterokaryons after a 4-hr incubation in either inhibitor. Both ouabain (L.M. Wiley, 1984, Dev. Biol. 105, 330-342) and phlorizin (this study) had a biphasic effect on the rate of nascent blastocoele fluid accumulation such that at lower concentrations (ouabain, 10(-5) M; phlorizin, 10(-6) M) fluid accumulation was accelerated and at higher concentrations (both inhibitors, 10(-4) M) fluid accumulation was delayed. In polar heterokaryons, both concentrations of each inhibitor caused the nuclei to become displaced basally from their normal location against the apical plasma membrane domain. Both nuclei, however, remained on the axis of polarity passing through the apical domain. The magnitude of displacement was greater at higher concentrations of either inhibitor. Neither inhibitor affected nuclear position in apolar heterokaryons. These observations agree with the hypothesis that apical plasma membrane solute transport maintains the asymmetric organelle distribution across the apical-basal axis of polar 16-cell-stage blastomeres.     

Occurrence and Ultrastructure of Collar Cells in the Stomach Gastrodermis of Polypodium hydriforme Ussov (Cnidaria)

The existence of collar cells lining the stomach gastrodermis in free-living Polypodium hydriforme and their ultrastructure are described. The collar cells are provided with a collar consisting of 9–10 microvilli which encircles a central flagellum and forms a flagellar pit. At the bottom of the pit around the basal part of the flagellum there is fine crystalline material which extends also in the spaces between the microvilli and keeps them straight. The flagellum has a typical axoneme (9+2), its basal body is located below the apical surface of the collar cell and continues into a striated rootlet. An accessory centriole is situated close to the upper part of the rootlet. The cell nucleus is located in the basal part of the cell. Prominent mitochondria with tubular cristae, Golgi cisternae and fragments of rough endoplasmic reticulum are situated mostly in the basal part of the cytoplasm. Discoidal vesicles are abundant in the apical cytoplasm. The collar cells are connected to each other by septate junctions and interdigitations. The ultrastructure of collar cells described here is discussed in comparison to that of other Cnidarians and in connection with the problem of Polypodium's systematic position.