Cation-distribution in developing follicles of the fruit fly Drosophila melanogaster

Abstract. Cations were precipitated with potassium antimonate in ovarian follicles of Drosophila and the distribution of the formed precipitates was studied. The precipitates were analyzed with a laser microprobe mass analyzer (LAMMA) and found to contain a high concentration of calcium; potassium and sodium were also detected. On counting the antimon precipitates in stage 10B follicles with the electron microscope, few precipitates per unit area were found in anterior nurse cells, but more in posterior nurse cells; the highest precipitate density occurred consistently in the oocyte. When follicles of different stages were compared, the precipitate density was found to increase in the ooplasm and in the posterior nurse cells during vitellogenesis, whereas it remained nearly constant in the anterior nurse cells. Thus, the ratio of precipitates between the posterior and anterior end of the follicle increases during vitellogenesis. It begins to decrease at the time when the nurse cells collapse. These results suggest that the electrical polarity observed in polytrophic ovarioles may be based on differences in the cation distribution along the antero-posterior axis of the follicle.     

Localized synthesis of specific proteins during oogenesis and early embryogenesis inDrosophila melanogaster

Summary Protein synthesis in egg follicles and blastoderm embryos ofDrosophila melanogaster has been studied by means of two-dimensional gel electrophoresis. Up to 400 polypeptide spots have been resolved on autoradiographs. Stage 10 follicles (for stages see King, 1970) were labelled in vitro for 10 to 60 min with³⁵S-methionine and cut with tungsten needles into an anterior fragment containing the nurse cells and a posterior fragment containing the oocyte and follicle cells. The nurse cells were found to synthesize a complex pattern of proteins. At least two proteins were detected only in nurse cells but not in the oocyte even after a one hour labelling period. Nurse cells isolated from stages 9, 10 and 12 follicles were shown to synthesize stage specific patterns of proteins. Several proteins are synthesized in posterior fragments of stage 10 follicles but not in anterior fragments. These proteins are only found in follicle cells. No oocyte specific proteins have been detected. Striking differences between the protein patterns of anterior and posterior fragments persist until the nurse cells degenerate. In mature stage 14 follicles, labelled in vivo, no significant differences in the protein patterns of isolated anterior and posterior fragments could be detected; this may be due to technical limitations. At the blastoderm stage localized synthesis of specific proteins becomes detectable again. When blastoderm embryos, labelled in vivo, are cut with tungsten needles and the cells are isolated from anterior and posterior halves, differences become apparent. The pole cells located at the posterior pole are highly active in protein synthesis and contribute several specific proteins which are found exclusively in the posterior region of the embryo. In this study synthesis of specific proteins could only be demonstrated at those developmental stages which are characterized by the presence of different cell types within the egg chamber, while no differences were detected when stage 14 follicles were cut and anterior and posterior fragments analyzed separately. The differences in the pattern of protein synthesis by pole cells and blastoderm cells indicate that even the earliest stages of determination are reflected by marked changes at the biochemical level.     

Follicle cell development is partly independent of germ-line cell differentiation in Drosophila oogenesis

Summary The developmental potential of the cells of the somatic follicular epithelium (follicle cells) was studied in mutants in which the differentiation of the germ-line cells is blocked at different stages of oogenesis. In two mutants, sn ^36a and kelch, nurse cell regression does not occur, yet the follicle cells around the small oocyte continue their normal developmental program and produce an egg shell with micropylar cone and often deformed operculum and respiratory appendages. Neither the influx of nurse cell cytoplasm into the oocyte nor the few follicle cells covering the nurse cells are apparently required for the formation of the egg shell. In the tumor mutant benign gonial cell neoplasm (bgcn) the follicle cells can also differentiate to some extent although the germ-line cells remain morphologically undifferentiated. Vitelline membrane material was synthesized by the follicle cells in some bgcn chambers and in rare cases a columnar epithelium, which resembled morphologically that of wild-type stage-9 follicles, formed around the follicle's posterior end. The normal polarity of the follicular epithelium that is characteristic for mid-vitellogenic stages may, therefore, be established in the absence of morphologically differentiating germ-line cells. However, the tumorous germ-line cells do not constitute a homogeneous cell population since in about 30% of the analyzed follicles a cell cluster at or near the posterior pole can be identified by virtue of its high number of concanavalin A binding sites. This molecular marker reveals an anteroposterior polarity of the tumorous chambers. In follicles mutant for both bgcn and the polarity gene dicephalic the cluster of concanavalin A-stained germ-line cells shifts to more anterior positions in the follicle.     

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.     

Ion currents and membrane domains in the cleaving Xenopus egg

We used an extracellular vibrating probe to measure ion currents through the cleaving Xenopus laevis egg. Measurements indicate sharp membrane heterogeneities. Current leaves the first cleavage furrow after new, unpigmented membrane is inserted. This outward current may be carried by K+ efflux. No direct involvement of the Na+,K+-ATPase in the generation of this outward current is detected at first cleavage. Inward current enters the old, pigmented membrane; however, it does not enter uniformly. The inward current is largest at the old membrane bordering the new membrane. This suggests a heterogeneous ion channel distribution within the old membrane. Experiments suggest that the inward current may be carried by Na+ influx, Ca2+ influx, and Cl- efflux. No steady currents were detected during grey crescent formation, the surface contraction waves preceding cleavage, or with groove formation at the beginning of cleavage.     

The microfilament pattern in the somatic follicle cells of mid-vitellogenic ovarian follicles of Drosophila

The microfilament pattern in the somatic follicle cells of mid-vitellogenic stage 9 to 11 follicles of Drosophila was analyzed by staining F-actin with fluorescence-labeled phalloidin. During the analyzed stages of oogenesis, the follicular epithelium differentiates morphologically and functionally. These changes are also reflected at the organization of the microfilaments. At stage 10, they show no preferred orientation in the very thin follicle cells covering the nurse cells. In contrast, the microfilaments in the basal part of the columnar follicle cells covering the oocyte become organized in parallel bundles oriented perpendicular to the long axis of the follicle. During stages 10B/11 this organization is maintained at the nurse cell/oocyte border but becomes more sloppy towards the posterior pole of the follicle. The basal part of the follicle cells containing the microfilament bundles adheres so tightly to the basement membrane that this acellular layer cannot be separated mechanically from the epithelium. Indirect evidence from inhibition studies with cytochalasins and the effects of collagenase or pronase E added to the culture medium suggest that the microfilament bundles may promote increased adhesiveness of the follicle cells to the basement membrane. The possible functional implications of the microfilaments and their orientation are discussed.     

Bioelectric aspects of the hemipteran telotrophic ovariole (Dysdercus intermedius)

Summary Two systems of steady extra-cellular currents were found along the surface of the telotrophicDysdercus ovarioles by means of a vibrating probe. The first covers the subgerminal tropharium and all the previtellogenic follicles. The current leaves the 3 or 4 small follicles of early euplasmic growth stages laterally and enters the syncytial tropharium. We presume that a similar intracellular current flows between the trophoplasm and the ooplasm which are interconnected by narrow nurse strands. Preliminary intracellular measurements indicate a potential gradient within this continuous cytoplasm, the ooplasm being electropositive to that of the tropharium. This current system fits into a model of polarized intracytoplasmic transport by electrophoresis. It is possible to explain the well known directed and selective flow of RNA from the tropharium via the nurse strands into the oocytes by means of such a model. The second current system occurs around every one of the 2 to 8 vitellogenic follicles. The pattern is completely different from that described for the first system. In the vitellogenic stages the current enters the follicle laterally all along the now much extended surface. It is balanced by a strong peak current which leaves the interfollicular region. As data on intracellular currents are not yet avialable, it is only a matter of speculation whether the circuit is closed through the ooplasm or only by a tangential loop through the follicle epithelium. The possible significance of this second current system for vitellogenin accumulation and uptake by the vitellogenic oocytes is also uncertain as yet.Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt Differenzierung)     

Distribution of ion channels in ascidian eggs and zygotes

Ascidian eggs and zygotes were whole-cell voltage-clamped and inward membrane currents, generated by stepping the membrane potential, studied from fertilization up to cytokinesis. Currents, induced by changing the voltage in steps from -80 to -30 mV, or to 0 mV, had maximum amplitudes which ranged from 400 to 1200 pA in the unfertilized egg and 100 to 1300 pA in the zygote. At 5 to 10 min after fertilization it was not possible to generate inward currents owing to the activity of nonspecific fertilization channels. Preceding cytokinesis, we observed a reduction in amplitude of the inward currents. By cutting eggs and zygotes into fragments, we have shown that the ion channels generating these inward currents are symmetrically distributed over the egg plasma membrane, but regionalized in the zygote with a maximum density at the animal pole.     

巨孔匙(虫戚)(Megathura)未受精卵细胞膜离子流的特征

用双微电极电压钳技术在巨孔匙(虫戚)(Megathura)未受精卵细胞膜上记录到多种离子流。主要有一种内向的两价离子流和几种钾离子流:包括钡离子激活的钾离子流,迅速激活又迅速失活的钾离子流(类似于I_A)和异常整流钾离子流。不同细胞的离子流大小不同。在一些卵可能会缺少其中某一种离子流。此外,还观察到浴槽溶液中氯和钠离子浓度改变对膜电位及膜电导的影响。     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Correlation of electrical current influx with nuclear position and division inFunaria caulonema tip cells

Summary Ionic currents around caulonema tip cells of the filamentous protonema of the mossFunaria hygrometrica were examined using a nonintrusive vibrating microelectrode to map electrical current before and during mitosis. Tip cells in interphase generate inward electrical currents that are maximal at the nuclear region. These currents remain concentrated over the nucleus as it migrates forward maintaining a constant distance from the growing tip. Just prior to mitosis this inward current increases twofold. During mitosis and cytokinesis current at the nuclear zone increases to four times the resting level and fluctuates, falling to zero after cell plate fusion with parental walls. The locus of outward current could not be dectected. These results suggest that plasma membrane ion currents may regulate both nuclear positioning and subsequent temporal and spatial control of cell division.     

Detection of extracellular calcium gradients with a calcium-specific vibrating electrode

We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase- sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.     

Development of a steady electric current in neonatal rat lumbrical muscle

We have used a vibrating probe and intracellular recording techniques to study the development of a steady electric current generated by rat lumbrical muscle. In adult animals, previous work has revealed a steady outward current generated at the end plate region. In the present study, we show that at birth muscles generate a steady inward, not outward current. The inward current declines with age, disappearing about 5 days after birth. At about the same time, the steady outward current appears, and reaches adult amplitude by 2-3 weeks after birth. The two currents are generated by completely different mechanisms. The inward current is blocked by alpha-bungarotoxin and apparently results from activation of acetylcholine-gated channels at the end plate. The outward current, on the other hand, is not affected by alpha-bungarotoxin but is blocked by agents which interfere with chloride movements across the membrane, as in the adult.     

Ion currents involved in early Nod factor response in Medicago sativa root hairs: a discontinuous single-electrode voltage-clamp study

Nod factor [NodRm-IV(Ac,S)], isolated from the bacterium Rhizobium meliloti, induces a well-known depolarization in Medicago sativa (cv Sitel) root hairs. Analysis of this membrane response using the discontinuous single-electrode voltage-clamp technique (dSEVC) shows that anion channel, K+ channel and H+-ATPase pump currents are involved in young growing root hairs. The early Nod-factor-induced depolarization is due to increase of the inward ion current and inhibition of the H+ pump. It involved an instantaneous inward anion current (IIAC) and/or a time-dependent inward K+ current (IRKC). These two ion currents are then down-regulated while the H+ pump is stimulated, allowing long-term rectification of the membrane potential (Em). Our results support the idea that the regulation of inward current plays a primary role in the Nod-factor-induced electrical response, the nature of the ions carried by these currents depending on the activated anion and/or K+ channels at the plasma membrane.     

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.     

Structural organization of ovarian follicle cells in the cotton bug (Dysdercus intermedius) and the honeybee (Apis mellifera)

Summary The somatic epithelia of Dysdercus and Apis follicles were analyzed by electron microscopy, and the patterns of F-actin and microtubules were studied by fluorescence microscopy. The epithelia in both species differ considerably in shape and in the organization of the cytoskeleton. During previtellogenic stages, the epithelium consists of columnar-shaped cells with small (Dysdercus) or no (Apis) lateral intercellular spaces. During vitellogenesis, the follicle cells round up; the intercellular spaces increase in size in Dysdercus follicles, whereas in Apis follicles they remain small. Along the basal surface of the follicle cells, there are conspicuous parallel bundles of microfilaments perpendicular to the anteroposterior axis of the follicles. In the honeybee, these microfilament bundles are present in long filopodia, most of which are embedded in thickenings of the basement membrane and extend over the surfaces of neighbouring cells. In the cotton bug, the basal surface of the follicle cells is thrown into parallel folds. The microfilament bundles are located just underneath the cell membrane where the folds contact the basement membrane. In the polar regions of the Dysdercus follicle, the epithelial cells become flat and adhere to each other without forming intercellular spaces. The basement membrane is particularly thick in the polar areas; this has also been observed in Apis follicles around the intercellular bridge connecting oocyte and nurse cells.     

Ionic mechanisms of depolarizing and hyperpolarizing quinine receptor potentials in Paramecium caudatum

The ionic mechanisms of the depolarizing and the hyperpolarizing quinine receptor potentials in the ciliate Paramecium caudatum were examined by using a behavioral mutant strain. The depolarizing receptor potential was induced by stimulating the anterior end of the specimen, and the hyperpolarizing receptor potential by stimulating the posterior end. The amplitude of both the depolarizing and the hyperpolarizing receptor potentials increased linearly with logarithmic increase in quinine concentration applied. Threshold concentration for inducing the depolarizing receptor potential was lower than that for the hyperpolarizing one. The peak level of the depolarizing receptor potential shifted towards the depolarizing direction with increasing external Ca²⁺ concentration while that of the hyperpolarizing receptor potential shifted in the depolarizing direction with increasing external K⁺ concentration. Under voltage-clamp conditions, the specimen produced an inward current in response to anterior stimulation, and an outward current in response to posterior stimulation. Both the peak inward and the peak outward currents showed a linear relationship with membrane potential. Current-voltage relationships of the receptor currents indicated conductance increase during the application of quinine. The depolarizing quinine receptor potential appears to be produced by an activation of Ca²⁺ channels, and the hyperpolarizing quinine receptor potential by an activation of K⁺ channels. Accepted: 3 October 1997     

The rôle of juvenile hormone in housefly ovarian follicle morphogenesis

Oögenesis in the housefly, Musca domestica, was divided into a series of 10 stages where stage 1 was the germarium, stage 4 was the beginning of yolk deposition, stage 7 was characterized by maximal nurse cell development, stage 9 by the degeneration of the nurse cells and chorion formation, and stage 10 was the mature egg. It required 69 hr from eclosion at 27°C to develop mature eggs. This represented an oöcyte volume increase of 3700-fold, a seventeenfold increase in follicle length, and a sevenfold increase in weight. The application of 2 μg of isopropyl (E,E)-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate (ZR-515) to allatectomized (-CA) flies stimulated egg development, which progressed at the same rate as the controls. The -CA flies did not develop eggs past stage 4, which represented a cessation of development at a volume of 1·4 per cent that of a mature egg and an ovarian dry weight of 11 per cent that of a mature ovary. The follicle cells from -CA flies did not differentiate into the squamous condition over the nurse chamber, did not become columnar over the oöcyte, did not produce the chorion or vitelline membrane, and did not decrease in number as they did on the stage 10 follicles. Endomitosis in the nurse cell nuclei of -CA flies stopped development at 290 c, but maximum development of 2400 c occurred in stage 7 follicles from controls, and then the nurse cells began to disintegrate.     

The histology and histochemistry of oogenesis in the water strider, Gerris remigis Say

In each ovariole of Gerris remigis, nurse cells arise by mitotic divisions at the anterior end of the germarium. These cells enlarge as they move posteriorly. This size increase is possibly caused by fusion of cells, but probably by endopolyploidy as well. The nurse cells then establish connections with a central trophic core, which receives the products of subsequent nurse cell degradation. Two possible pathways of nuclear degradation are suggested: one involves the condensation of chromatin within the nucleus; the other, the release of DNA as fine granules into the cytoplasm. Cytoplasmic areas containing such DNA are also rich in proteinaceous granules, but have a meager content of RNA. The remainder of the cytoplasm of the mature nurse cells contains a high concentration of RNA, as do the nucleoli. Posteriorly the trophic core connects via nutritive cords with each developing oocyte in the prefollicular region and in the anterior vitellarium. RNA is apparently contributed to the ooplasm via the trophic stream. Patches of cytoplasmic DNA are present in the young oocytes; the origin and fate of this DNA is uncertain. During early oocyte maturation chromosomal stainability decreases, and the nucleolus enlarges. In previtellogenic stages, numerous proteinaceous bodies appear in association with the nucleolus-chromosome complex. These bodies, like the nucleolus, have only a low RNA content. They may pass to the cytoplasm, but cannot be traced with certainty. During the latter part of this period a complex population of small proteinaceous and lipid preyolk bodies accumulates peripherally in the oocyte. Definitive protein and lipid yolk are probably derived by the enlargement and inward migration of these bodies. The oocytes are each surrounded by a layer of follicle cells proliferated in the prefollicular region. These become binucleate and enlarge as the enclosed oocytes grow and elongate. RNA also increases in the nucleoli and cytoplasm of the follicle cells as they move posteriorly in the vitellarium. There is no evidence of transfer of nucleic acids or protein from the follicle cells to the oocyte. The nurse cells are therefore implicated as the major source of nucleic acids for the maturing oocyte.     

Dicephalic — ADrosophila mutant affecting polarity in follicle organization and embryonic patterning

Summary The mutationdicephalic (dic) affects follicle development and thereby alters the antero-posterior polarity of embryonic patterning. It maps at a single locus (3–46.0±1.0) and can be characterized as a semi-dominant maternal effect mutation with low penetrance. Indic follicles, the 15 nurse cells form two clusters located at opposite poles of the oocyte; the numerical distribution of the nurse cells among the clusters varies from 7:8 to 1:14. Thedic egg shell carries a micropyle (anterior marker) at either pole, but the misshapen respiratory appendages are restricted to one of the two poles in most eggs. The malformed eggs rarely yield larvae and these are always abnormal anteriorly and/or posteriorly. The segment pattern expressed in their cuticle may represent two anterior parts of opposite polarities (double head type), two posterior parts of opposite polarities (double abdomen type, rare) or show uniform polarity. Lability of organization at the cystocyte stage appears as the primary developmental defect of the mutant.