Electrically mediated protein movement inDrosophila follicles

Summary Distribution of rhodamine-conjugated lysozyme injected into the sixteen-cell syncytium comprising the germ-line portion of theDrosophila follicle is shown to be affected by charge. Positive molecules are able to migrate through intercellular bridges from the oocyte to the nurse cells, but are unable to migrate detectably from nurse cells to the oocyte. Their negatively charged counterparts can move from the nurse cells to the oocyte, but are unable to traverse the intercellular bridges in the counter direction. This charge-dependent movement of molecules is accompanied by an electrical potential difference, focused across the nurse cell-oocyte bridges, which makes the nurse cells negatively charged to the oocyte. The addition of insect hemolymph to the physiological salt solution in which the experiments were performed resulted in only a small increase in the transmembrane resistance, but enhanced the potential difference between oocyte and nurse cells from 0.2±0.3 (SE) mV (nurse cells negative) to 2.3±0.45 (SE) mV (nurse cells negative). Supported by NSF Grant # DB-18617     

Yolk formation in Isohypsibius (Eutardigrada)

Summary In Isohypsibius granulifer, yolk is autosynthesized. The Golgi apparatus is mainly responsible for the formation of yolk, which consists of irregular platelets with heterogeneous contents and a diameter of about 1 m. Dense globules, 300 nm in diameter, are visible among yolk platelets. These develop in the vesicles of the rough endoplasmic reticulum. The genesis of these vesicles is associated with the outer membrane of the nuclear envelope, which forms blebs intensively during previtellogenesis and early vitellogenesis. The developing oocytes are assisted by nurse cells, to which they are jointed by cytoplasmic bridges. For every oocyte, there are a number nurse cells, which are sister cells of the oocyte. In addition to rRNA, nurse cells transfer to the oocyte lipids, platelets of yolk formed in their cytoplasm, mitochondria and cortical granules.     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary The first stages of the oogenesis of Triops cancriformis have been studied. At the outset the oocyte is smaller than the nurse cells. Meiosis begins with typical synaptonemal complexes. The significance of these complexes and of some other peculiar structures of germ cells, i.e., pore complexes and annuli within the nucleus, and annulate lamellae within the cytoplasm are discussed. The morphofunctional uniformity of some cytoplasmic structures (annulate lamellae, concentrically arranged ER, and yolk globules) in the oocyte as well as its nurse cells is also discussed.     

Development of the oocyte and its accessory cells of the polychaete,Diopatra cuprea (Bosc)

The oocyte-nurse cell complex of the polychaetous annelid, Diopatra cuprea, has been explored by various methods of light microscopy and by the technique of electron microscopy. Early in its development the complex appears as a string of cells floating within the coelomic cavity. As this string of cells develops, the volume of one cell (approximately the middle one) increases greatly; while that of the remaining cells, referred to as nurse cells, increase slightly. Due to this differential growth, the two opposing strands of nurse cells are displaced to one side of the oocyte. Nurse cells are joined to one another by cytoplasmic bridges. Cytoplasmic bridges also exist between the strands of nurse cells and the oocyte. The presence of numerous ribosomes within the bridges between the oocyte and nurse cells encourages us to suggest that this organelle may be transferred to the oocyte via this route. The transported ribosomes may be used by the maturing oocyte, or they may be stored by the egg to be utilized during embryogenesis. Moreover, we believe that the nurse cells are not involved in the production of the protein-carbohydrate yolk bodies for we think that these are elaborated by the endoplasmic reticulum in collaboration with certain Golgi complexes of the oocyte.     

Observations on the polarity of mutant Drosophila follicles lacking the oocyte

Summary Homozygous females of the mutantsegalitarian andBicaudal-D ^R26produce follicles in which the oocyte is replaced by an additional nurse cell. Normal morphological markers for polarity can be identified in mutant follicles but the normal spatial organization of these markers is disturbed. For example, nurse-cell nuclei of different ploidy classes are present but, contrary to wild-type follicles, the nuclei show no anteroposterior ploidy gradient. The two cells with four intercellular bridges, one of which should have developed into the oocyte rather than a nurse cell, are located at the posterior pole only in young follicles (up to about stage 5), whereas during later stages they are more often found at lateral or intermediate positions. This disturbed polarity correlates with a variable aberrant pattern of extracellular ionic currents. Moreover, in the mutant follicles patches of columnar follicular epithelium differentiate locally although this type of epithelium forms normally only around the oocyte. The follicle cells at both follicle poles possess anterior quality since they migrate from both poles towards the centre of the follicle, as do the border cells restricted to the anterior pole in wild-type follicles. Our analysis indicates that in the mutants the follicular polarity is normal at first but becomes disturbed during stages 5 to 6. The secondary breakdown of polarity is likely to follow on from the absence of the oocyte.     

Studies on the ovotestis of the slug Agriolimax reticulatus (Müller)

Summary The follicle cells, nurse cells and germinal epithelia, which are closely associated with the oocyte of Agriolimax reticulatus (Müller) during its development in the ovotestis, have been studied using light and electron microscopy. The various secretory, digestive and phagocytic activities of these cells have also been investigated using electron cytochemical tests for oxidisable polysaccharide, acid phosphatase and electron-opaque tracer molecules. The oocyte lies initially between the germinal epithelia and a layer of nurse cells but, as oocyte vitellogenesis proceeds, it becomes encapsulated by a layer of follicle cells. Both the follicle and the nurse cells are active in secretion and digestion and contain Golgi apparatus, granular endoplasmic reticulum and acid phosphatase-rich digestive vacuoles. The significance of these activities is discussed in relation to oocyte vitellogenesis, secondary envelope formation and the digestion and recycling of cellular material.     

Oogenesis in the honeybee Apis mellifera: cytological observations on the formation and differentiation of previtellogenic ovarian follicles

Summary Oogenesis is known to be important for embryonic pattern formation. For this reason we have studied the early differentiation of the honeybee ovariole histologically, ultrastructurally, and by staining F-actin with rhodaminyl-phalloidin. At the anterior tip of the ovariole, stem cells are lined up in a single file; they are organelle-poor but contain characteristic electrondense bodies with lysosomal properties. The presence of these bodies in cystocytes as well as prefollicle cells indicates that both cell types may be derived from the apical stem cells. During later stages of oogenesis, the follicle cells differentiate cytologically in different regions of the follicle. The organization of the intercellular bridges between cystocytes derived from a single cystoblast has been studied in detail. The polyfusomes in the intercellular bridges of cystocyte clusters stain with rhodaminyl-phalloidin and hence contain F-actin. Later, when the polyfusomes begin to desintegrate, F-actin rings form which line the rims of the intercellular bridges. Actin might be recruited from conspicuous F-actin stores which were detected in the germ-line cells. The F-actin rings are dissembled some time before the onset of vitellogenesis when the nurse chamber has grown to a length of about 200 m. At the basal side of the follicle cells (close to the basement membrane facing the haemocdele) parallel microfilament bundles encircle the ovariole. The microfilament bundles which are oriented mostly perpendicular to the long axis of the ovariole were first observed around the zone where the cystocyte divisions occur; after this phase the micro-filament bundles become organized differently in the follicle cells associated with the nurse cells and in the follicular epithelium of the oocyte. Correspondence to: H.O. Gutzeit     

Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis

We expressed two green fluorescent protein (GFP)-tagged Nopp140 isoforms in transgenic Drosophila melanogaster to study nucleolar dynamics during oogenesis and early embryogenesis. Specifically, we wanted to test whether the quiescent oocyte nucleus stored maternal Nopp140 and then to determine precisely when nucleoli formed during embryogenesis. During oogenesis nurse cell nucleoli accumulated GFP-Nopp140 gradually such that posterior nurse cell nucleoli in egg chambers at stage 10 were usually brighter than the more anterior nurse cell nucleoli. Nucleoli within apoptotic nurse cells disassembled in stages 12 and 13, but not all GFP-Nopp140 entered the oocyte through inter-connecting cytoplasmic bridges. Oocytes, on the other hand, lost their nucleoli by stage 3, but GFP-Nopp140 gradually accumulated in oocyte nuclei during stages 8–13. Most oocyte nuclei at stage 10 stored GFP-Nopp140 uniformly, but many stage 10 oocytes accumulated GFP-Nopp140 in presumed endobodies or in multiple smaller spheres. All oocyte nuclei at stages 11-12 were uniformly labeled, and GFP-Nopp140 diffused to the cytoplasm upon nuclear disassembly in stage 13. GFP-Nopp140 reappeared during embryogenesis; initial nucleologenesis occurred in peripheral somatic nuclei during embryonic stage 13, one stage earlier than reported previously. These GFP-Nopp140-containing foci disassembled at the 13th syncytial mitosis, and a second nucleologenesis occurred in early stage 14. The resulting nucleoli occupied nuclear regions closest to the periphery of the embryos. Pole cells contained GFP-Nopp140 during the syncytial embryonic stages, but their nucleologenesis started at gastrulation. This work was supported by the National Science Foundation (grant MCB-0234245). O'Keith Dellafosse was supported by the Louisiana Alliance for Minority Participation (LAMP).     

Aberrant oogenesis in the patterning mutant dicephalic of Drosophila melanogaster: time-lapse recordings and volumetry in vitro

Summary Drosophila females homozygous for the mutation dicephalic occasionally produce ovarian follicles with a nurse-cell cluster on each oocyte pole (dic follicles). Most dic follicles contain 15 nurse cells as in the normal follicle, but the total nurse-cell volume is larger in dic follicles; this is in keeping with the increase in DNA content recently described. However, the relative increase in oocyte volume during nurse-cell regression (from stage 10B onward) is not significantly larger in dic than in normal follicles. Time-lapse recordings in vitro show that, as a rule, both nurse cell clusters in a dic follicle export cytoplasm to the oocyte but nurse-cell regression remains incomplete at both poles and the persisting remnants of the nurse cells cause anomalies in chorion shape. The kinematics of cytoplasmic transfer are less aberrant at that oocyte pole which harbours the germinal vesicle. Possible links are discussed between these anomalies of oogenesis and the double-anterior embryonic patterns observed in the majority of developing dic eggs.     

Mutations in supernova,indicate that this gene is required for the division of germ line cells in Drosophila

Mutations in supernova, previously shown to uncouple chromosome replication from segregation during cleavage in Drosophila embryos, also sanctions extra divisions of cystoblasts and spermatoblasts. This leads either to the formation of egg chambers which contain more than fifteen nurse cells or testes which have an excess of spermatocytes. In maturing egg chambers two potential oocytes may be specified in which case they are often ectopically located and connected with surrounding nurse cells by four ring canals. However, a typical oocyte nucleus is not always present and these chambers usually become necrotic and degenerate. The nurse cells are of variable size, but are still interconnected by a system of ring canals. They all possess a polyploid nucleus. Sequestering of maternal mRNA's from the nurse cells into the potential oocyte(s) takes place but there is no localization of this maternal information within the oocyte probably because of defective microtubule assembly. Many spermatocytes fail to complete meiosis so that bundles of spermatids are reduced in size and the males have reduced fertility. It is proposed that this gene is indirectly involved in regulating the timing of mitotic divisions in both cystoblasts and spermatoblasts through its interference with microtubule assembly which is consistent with its role during embryogenesis.     

Steady-state gradient in calcium ion activity across the intercellular bridges connecting oocytes and nurse cells in Hyalophora cecropia

Intracellular activities of K⁺, H⁺, Mg²⁺, Ca²⁺, and Cl^?, measured with ion selective microelectrodes in the oocyte and the nurse cells in ovarian follicles of Hyalophora cecropia, indicated that a Ca²⁺ current is a key component of the electrical potential that is maintained across the intercellular bridges connecting these two cells. In vitellogenic follicles, Ca²⁺ activity averaged 650 nM in the oocyte and 190 nM in the nurse cells, whereas activities of the other ions studied differed between these cells by no more than 6%. Incubation in 200 μM ammonium vanadate caused a reversal of electrical potential from 8.3 mV, nurse cell negative, to 3.0 mV, oocyte negative, and at the same time the Ca²⁺ gradient was reversed: activities rose to an average 3.0 μM in the nurse cells and 1.6 μM in the oocyte, whereas transbridge ratios of the other cations remained at 0–3%. In immature follicles that had not yet initiated their transbridge potentials, Ca²⁺ activities averaged ～? 2 μM in both oocyte and nurse cells. The results suggest that vitellogenic follicles possess a vanadatesensitive Ca²⁺ extrusion mechanism that is more powerful in the nurse cells than in the oocyte. © 1994 Wiley-Liss, Inc.     

Vitellogenic ovarian follicles of Drosophila exhibit a charge-dependent distribution of endogenous soluble proteins

In ovarian follicles of Drosophila, soluble endogenous charged proteins are asymmetrically distributed dependent upon their ionic charge. Reversal of the normal ionic difference across the intercellular bridges which connect nurse cells to their oocyte results in a redistribution of these proteins. Twelve soluble endogenous acidic proteins were identified by 2-D gel electrophoresis as being present in both oocytes and nurse cells in samples run on four or more gels. Of these, following osmotically induced reversal of the electrical transbridge gradient the concentration of seven proteins decreased in the oocyte while nurse cell concentrations of all twelve proteins increased. Of seven basic proteins analyzed, following reversal of the electrical gradient the concentration of all seven increased in oocytes. Four of these decreased in nurse cells, while nurse cell concentrations of the remaining three basic proteins also appeared to decrease, but yielded spots too faint for measurement. Data presented here demonstrate that, as in the Saturniidae, the ionic gradient across the nurse cell-oocyte intercellular bridges of the dipteran, Drosophila, can influence the distribution of soluble endogenous charged molecules.     

POLARIZED INTERCELLULAR BRIDGES IN OVARIAN FOLLICLES OF THE CECROPIA MOTH

Fluorescein-labeled rabbit serum globulin was injected into vitellogenic oocytes of the cecropia moth. Though the label spread throughout the ooplasm in less than 30 min, it was unable even after 2 h to cross the complex of intercellular bridges connecting the oocyte to its seven nurse cells. After injection into a single nurse cell, fluorescence was detected in the oocyte adjacent to the bridge complex within 3 min and had spread throughout the ooplasm in 30 min. Here also, the cell bodies of the six uninjected nurse cells remained nonfluorescent. Four of the nurse cells are not bridged directly to the oocyte but only through the apical ends of their siblings. Unidirectional movement must therefore occur in the apical cytoplasm of the nurse cells, as well as in the intercellular bridges. The nurse cells of healthy follicles had an intracellular electrical potential -40 mV relative to blood or dissecting solution, while oocytes measured -30 mV. A mV difference was also detected by direct comparison between a ground electrode in one cell and a recording electrode in the other. Three conditions were found in which the 10 mV difference was reduced or reversed in polarity. In all three cases fluorescent globulin was able in some degree to cross the bridges from the oocyte to the nurse cells.     

INTERCELLULAR MIGRATION OF CENTRIOLES IN THE GERMARIUM OF DROSOPHILA MELANOGASTER : An Electron Microscopic Study

A cluster of centrioles has been found in the early Drosophila oocyte. Since the oocyte is connected to 15 nurse cells by a system of intercellular bridges or ring canals, the possibility that the cluster of centrioles arose in the germarium from an intercellular migration of centrioles from the nurse cells to the oocyte was analyzed in serial sections for the electron microscope. Initially, all of the 16 cells of the future egg chambers possess centrioles, which are located in a juxtanuclear position. At the time the 16 cell cluster becomes arranged in a lens-shaped layer laterally across the germarium, the centrioles lose their juxtanuclear position and move towards the oocyte. By the time the 16 cell cluster of cells is surrounded by follicle cells (Stage 1), between 14 and 17 centrioles are found in the oocyte. Later, these centrioles become located between the oocyte nucleus and the follicle cell border and become aggregated into a cluster less than 1.5 µ in its largest dimension. The fate of these centrioles in the oocyte is not known. The fine structure of the germarium and the early oocyte is also described.     

Ovarian Structure in Milnesium tardigradum (Tardigrada, Milnesiidae) during Early Vitellogenesis

The ultrastructure of the ovary of Milnesium tardigradum during early vitellogenesis is described. Within the ovary, there were large multinuclear cells surrounded by many mononuclear oocytes. Observation of serial sections revealed four multinuclear cells that were connected to each other by cytoplasmic bridges. Each peripheral oocyte was connected to the multinuclear cell. An enormous ER-like structure was conspicuous in the centre of the multinuclear cell. The presence of large numbers of lipid droplets and yolk granules in both multinuclear cells and many mononuclear oocytes suggested a role as nurse cells. A small number of these oocytes grow to be eggs. The structural features of the multinuclear nurse cell were compared with other known examples.     

Cellular and molecular markers of anteroposterior and dorsoventral organisation in the vitellogenic follicles of adult Sarcophaga bullata (Diptera) and dorsoventral orientation of follicles in the ovary

Summary Polar organisation in the follicles of adult Sarcophaga bullata is reflected in the nurse cell-oocyte axis and in the orientation of the two polar cell pairs in the follicular epithelium. The internal organisation of the nurse cell chamber contributes to polarity but not to dorsoventral asymmetry. Dorsoventral asymmetry is correlated with the eccentric position of the germinal vesicle and the orientation of the polar cell pairs; no other follicle cell specialisations are seen. In an ovary, follicles are preferentially orientated with the dorsal side to the centre of the ovary. Cytoskeletal and some haemolymph proteins are molecular markers of polarity. Thus, in pre-vitellogenic stages, tubulin immunoreactivity is higher in the oocyte than in the nurse cells, actin immunoreactivity is the same over the cystocytes and larval serum proteins are restricted to the poles. During vitellogenesis, both actin and tubulin become more concentrated in the nurse cells and larval serum protein 1 accumulated in the polar cells during border cell migration when yolk polypeptides also accumulate in the oocyte. At the end of vitellogenesis a lipophorin is taken up by the oocyte. No molecular marker of dorsoventral asymmetry was identified.     

Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport.

Inhibitor studies have implicated microtubules in at least three important developmental processes during Drosophila oogenesis: oocyte determination and growth during stages 1 through 6, positioning of the anterior determinant bicoid mRNA during stages 9 through 12, and ooplasmic streaming during stages 10b through 12. We have used fluorescence cytochemistry together with laser scanning confocal microscopy to identify distinct microtubule structures at each of the above three periods that are likely to be involved in these processes. During stages 1 through 7, maternal components synthesized in nurse cells are transported through cytoplasmic bridges to the oocyte. At this time, microtubules that appear to originate in the oocyte pass through these cytoplasmic bridges into the adjacent nurse cells; these microtubules are likely to serve as a polarized scaffold on which maternal RNAs and proteins are transported. During stages 7 and 8, microtubules in the oocyte cortex reorganize to form an anterior-to-posterior gradient, suggesting a role for microtubules in the localization of morphogenetic determinants. Finally, when ooplasmic streaming begins during stage 10 b, it is accompanied by the assembly of subsurface microtubule arrays that spiral around the oocyte; these arrays disassemble as the oocyte matures and streaming stops. During ooplasmic streaming, many vesicles are closely associated with the subsurface microtubules, suggesting that streaming is driven by vesicle translocation along microtubules. We believe that actin plays a secondary role in each of these morphogenetic events, based on our parallel studies of actin organization during each of the above stages of oogenesis.     

A Dual Role for Actin and Microtubule Cytoskeleton in the Transport of Golgi Units from the Nurse Cells to the Oocyte Across Ring Canals

Axis specification during Drosophila embryonic development requires transfer of maternal components during oogenesis from nurse cells (NCs) into the oocyte through cytoplasmic bridges. We found that the asymmetrical distribution of Golgi, between nurse cells and the oocyte, is sustained by an active transport process. We have characterized actin basket structures that asymmetrically cap the NC side of Ring canals (RCs) connecting the oocyte. Our results suggest that these actin baskets structurally support transport mechanisms of RC transit. In addition, our tracking analysis indicates that Golgi are actively transported to the oocyte rather than diffusing. We observed that RC transit is microtubule-based and mediated at least by dynein. Finally, we show that actin networks may be involved in RC crossing through a myosin II step process, as well as in dispatching Golgi units inside the oocyte subcompartments.