The Localisation of ATP in the Polar Lobe of the IIyanassa Egg

The ATP content of the Illyanassa egg, the lobeless egg and the third polar lobe was measured. The third polar lobe contained 37.8 per cent of the ATP of the intact egg.     

Organogenesis in normal and lobeless embryos of the marine prosobranch gastropod Ilyanassa obsoleta

The histogenetic and organogenetic capabilities of normal and lobeless embryos of Ilyanassa obsoleta are analyzed. Larvae developed from eggs in which the polar lobe was removed at the trefoil stage of first cleavage were studied in wholemount and section. None showed any evidence of eyes, statocysts, operculum, external shell, heart, or intestine. Sixty per cent of the wholemount larvae observed under polarized light exhibited internal masses of birefringent material resembling shell material. Of eight- to nine-day lobeless larvae studied as stained, sectioned material 100% possessed muscle tissue; 97.5% possessed velar tissue, digestive gland tissue, and style sac tissue; 52.5% possessed two or more style sac areas; 71% exhibited esophagus; 63% possessed stomach tissue; 37% exhibited stomodeal-like invaginations of the ectoderm; 31% exhibited a lightly staining ciliated area at the tip of the prevelar ectoderm; and 21% possessed glandular ectoderm resembling pedal or mantle gland tissue. The evidence presented suggests that the material of the polar lobe has an important role not only in cellular differentiation but also in the organization of tissue types into organs or organ primordia.     

Role of the Vegetal Body in the Regulation of Development in Bithynia tentaculata (Prosobranchia, Gastropoda)

In the first polar lobe of the egg of Bithyma tentaculata thereis a cup-shaped mass of small vesicles rich in RNA, called thevegetal body. It is passed to the CD blastomere with the polarlobe and then its contents are distributed to C and D. Whenthe polar lobe is removed, embryos form neither mesentoblastnor mesoderm bands. They fail to establish bilateral symmetryor to form eyes, foot, operculum and shell. AB half embryosdevelop only the structures found in lobeless embryos, whileCD halves develop lobe dependent adult structures such as eyes,foot and shell. AD- and BC-2/4-embryos develop similarly andboth have lobe dependent structures. Three quarter embryos developas in other molluscs except that ABD (–C) develops poorlyand ABC (–D) develops all lobe-dependent structures. Thus,all evidence available indicates that C receives cytoplasm usuallyrestricted to D in molluscs and therefore has the capacity toexert a similar morphogenetic influence on development.     

Shell differentiation and engrailed expression in the Ilyanassa embryo

 We have used in situ hybridization and immunocytochemistry to study the expression of the engrailed-related gene, Ily-en in embryos of the marine mud snail Ilyanassa obsoleta. We find that Ily-en is only expressed in shell gland cells. Only mRNAs localized in the shell gland hybridize to an antisense probe of the Ily-en homeobox. Similarly, only shell gland cells or shell-forming cells are stained by the monoclonal antibody 4D9, which was raised to the engrailed-class protein from Drosophila. Ilyanassa embryos made deficient in vegetal cytoplasm by removing the third polar lobe fail to differentiate an organized external shell. They do however make some randomly oriented internal shell fragments in which Ily-en is expressed. Because Ily-en is expressed in shell gland cells of both normal and lobeless embryos, we conclude that the determinant(s) required for Ily-en expression are not exclusively localized in the polar lobe. Received: 16 October 1997 / Accepted: 9 January 1998     

The role of unequal cleavage and the polar lobe in the segregation of developmental potential during first cleavage in the embryo of Chætopterus variopedatus

Summary The inequality of the first cleavage division of the Chætopterus embryo is caused by the production of a small polar lobe and the internal shifting of the first cleavage spindle. This division produces a two-celled embryo containing a small AB and a large CD blastomere. These blastomeres have different morphogenetic potentials. Only the larvae resulting from isolated CD blastomeres are able to form bioluminescent photocytes, eyes and lateral hooked bristles. The removal of the polar lobe during first cleavage does not have a great effect on development. These lobeless embryos display a normal pattern of cleavages through the time of mesentoblast formation. The resulting larvae are essentially normal, however they do not form functional photocytes. If the CD cell is isolated after the removal of the first polar lobe, the resulting larva is virtually identical to those formed by the intact CD cell except it lacks the photocyte cells. These results indicate that two separate pathways are involved in the segregation of developmental or morphogenetic potential which takes place during first cleavage. One set of factors, which are necessary for photocyte formation, are associated with the first polar lobe. Other factors that are necessary for the formation of the eyes and lateral hooked bristles are segregated by the unequal cleavage which results from an internal shifting of the cleavage spindle. The removal of a large portion of the vegetal region of the embryo during first cleavage leads to the production of larvae which display a decreased ability to form eyes and lateral hooked bristles. These embryos frequently display an abnormal pattern of cleavages. They do not form the primary somatoblast or the mesentoblast. These results indicate that the vegetal region of the CD cell of Chætopterus is analogous to polar lobes which have been studied in other species, and is therefore important in the specification of the D quadrant. These features of the first cleavage of Chætopterus are a combination of those displayed by forms with direct unequal cleavage and other forms which cleave unequally through the production of large polar lobes. The significance of these findings is discussed relative to the origins of these different types of unequal cleavage.     

Effect of Ca2+ on deformation, polar body extrusion and pronucleus formation in the egg of the ascidian, Ciona savignyi

Cortical deformation and polar body extrusion are the principal events that occur at fertilization in the ascidian egg. We demonstrated that the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the fertilized egg of Ciona savignyi increased at egg deformation (main peak) and then several small Ca²⁺ spikes (1st spikes) appeared before the first polar body extrusion. Brief Ca²⁺ spikes (2nd spikes), then appeared in the period between the first and second polar body extrusion. When eggs were fertilized in Ca²⁺-free artificial seawater, the main peak and 1st spikes appeared, but the 2nd spikes did not, suggesting that the Ca²⁺ required for the main peak and 1st spikes is released from the intracellular store in this species and that extracellular Ca²⁺ is required for the 2nd spikes. When [Ca²⁺]_i was clamped at a low level (0.03–0.13 μmol/L) by injecting the egg with low-Ca²⁺ buffers and the egg was then inseminated, deformation, polar body extrusion and pronucleus formation were suppressed. In contrast, egg deformation and first polar body extrusion were induced without insemination when [Ca²⁺]_i was 0.9 μmol/L. A higher Ca²⁺ concentration of 1.2–10.1 μmol/L was required for extrusion of the second polar body and pronucleus formation. These data suggest that sequential Ca²⁺ increases (i.e. main peak and 1st and 2nd spikes) are prerequisite for the deformation and polar body extrusion of the egg. Furthermore, in eggs arrested at the second meiotic metaphase after first polar body extrusion by the injection of Ca²⁺ buffer, subsequent injection of excess Ca²⁺ caused formation of an irregular second polar body-like protrusion, suggesting latent arrest at the second meiotic metaphase in the ascidian egg.     

Nucleic acid synthesis in the normal and lobeless embryo of Ilyanassa obsoleta.

The RNA and DNA content of the normal and lobeless embryo of the Ilyanassa embryo was measured at several stages of embryogenesis. In the lobeless embryo a delay in the net accumulation of RNA and a decrease in the rate of DNA synthesis were observed. It is suggested that the failure of some of the lobe dependent organs to differentiate may be caused by a reduced rate of proliferation of determined embryonic cells.     

Significance of the polar lobe for the determination of dorsoventral polarity in Dentalium vulgare (da Costa)

The development of dorsoventral polarity in Dentalium dentale has been analyzed after inhibiting first polar lobe formation with cytochalasin B and bisecting the egg into two equal parts at an early trefoil stage. Cleavage pattern and morphogenesis have been studied in both in vivo and permanent cytological preparations. After bisecting the egg, each blastomere may fuse with its adhering polar lobe half and subsequently behave as a CD blastomere. The polar lobe substance may induce both halves to develop an apical tuft and probably also a posttrochal region. Cytochalasin B embryos which pass through an equal first cleavage form a four-cell stage in which the two D blastomeres are situated opposite or adjacent to each other (CDCD or CCDD embryos, respectively). During further development the larvae show a duplication of lobe-dependent structures. It is concluded that dorsoventral polarity originates epigenetically by fusion of the polar lobe with one of the first two blastomeres and is not preformed in the uncleaved egg.     

An unusual cytoplasmic organelle in oocytes of Ilyanassa obsoleta

An unusual organelle in the oocytes of the marine mud snail Ilyanassa obsoleta is described and called a polymerosome, because of its several components. Polymerosomes appear at the beginning of vitellogenesis and persist, in part, through postvitellogenesis. The persistent part is similar to an organelle that has been described by other workers in the vegetal region and the polar lobe of the Ilyanassa egg.     

Composition of the nucleotides pool in a morphogenetic compartment in eggs of Nassarius reticulatus (mollusca) analysed by capillary isotachophoresis

The spectrum of low molecular weight compounds, in particular of ribonucleotides, within first cleavage stage embryos of the polar lobe-forming mollusc Nassarius reticulatus and the distribution of the compounds within the embryo at the trefoil stage of first cleavage are analysed by means of capillary isotachophoresis after 0.5 M PCA extraction. The compounds which are found in the whole trefoil embryo (T), the lobeless part (LL), and the polar lobe (PL) respectively, and the mean quantities (nmol. microliter-1; n = 6) are: UTP (11.5, 4.8, 5.6), ITP (8.5, 3.6, 5.0), GTP (10.3, 3.0, 9.0), ATP (29.8, 13.4, 18.8), UDP (11.8, 3.4, 8.7), CTP (8.0, 3.1, 4.5), GDP (5.3, 2.6, 3.4), ADP (16.5, 6.1, 11.6), CDP (4.0, 1.4, 2.6), GMP (4.7, 2.7, 4.3), glucose-6-phosphate (G6P) (53.5, 38.8, 13.0). These compounds appear to be localized in the non-yolk cytoplasmic pool. As the volume ratio of PL/LL for total volume and for non-yolk cytoplasmic volume is about 0.74 and 0.60 respectively, the concentration of all nucleotides in PL as compared to LL is significantly higher (HO, p less than 0.001), both relative to the total volume and to the non-yolk cytoplasmic volume. The G6P concentration is considerably higher in the lobeless part. The morphogenetic role of the vegetal pole compartment of the egg apparently is correlated with a relatively high level of its nucleotide contents.     

Lactobacillus gasseri PA-3, but not L. gasseri OLL2996, reduces the absorption of purine nucleosides in rats

Lactobacillus gasseri PA-3 (PA-3) is a bacterial strain with a strong ability to degrade purine nucleosides. We previously showed that PA-3 incorporates purines in vitro and that oral administration of PA-3 and purines to rats attenuated their absorption of purines. It remains unclear whether these effects of PA-3 depend on bacterial strains. This study therefore compared the abilities of PA-3 and another bacterial strain of L. gasseri, OLL2996, which has shown decreased ability to degrade purine nucleosides in vitro, to incorporate purine nucleosides and to inhibit the absorption of purines fed to rats. Each bacterial strain was incubated in the presence of ¹⁴C-adenosine or ¹⁴C-inosine and the incorporation of each purine was evaluated by measuring their radioactivity. In vivo, rats were fed ¹⁴C-labeled purines along with PA-3 or OLL2996 and the absorption of these ¹⁴C-labeled purines was evaluated by analyzing radioactivity of blood samples. PA-3 incorporated about twice as much ¹⁴C-adenosine and ¹⁴C-inosine as OLL2996. The elevation of radioactivity levels in blood was 10–20% lower in rats treated with PA-3 than in control rats, after feeding with both ¹⁴C-adenosine and ¹⁴C-inosine as purines. In contrast, treatment with OLL2996 did not have statistically significant effects on radioactivity compared with the control group. These results indicate that the magnitude of bacterial inhibition of purine absorption is dependent on bacterial strain, correlating at least partly with the ability to incorporate and degrade purines.     

Effects of local anesthetics on cytokinesis and polar lobe formation in fertilized eggs of Ilyanassa obsoleta

Summary

We have examined the ability of fertilized eggs of Ilyanassa obsoleta to form polar lobe constrictions and undergo cytokinesis in the presence of several local anesthetics and compared these effects with those of drugs known to affect microtubules. Procaine, lidocaine (Xylocaine), mepivacaine, tetracaine, and dibucaine all delay the beginning of polar lobe constrictions at low concentrations and in the order of their lipid solubilities. All of the anesthetics are effective at lower concentrations in the absence of extracellular Ca²⁺. Procaine and tetracaine ‘lock’ cells for several hours halfway through the constriction of the polar lobe neck and prevent subsequent cytokinesis, effects similar to those of the microtubule agents, colchicine and nocodazole. Procaine has no effect on membrane potential, ψ_m, or on intracellular chloride activity, (Cl)_c, as determined with ion-selective microelectrodes. This suggests that procaine does not inhibit cellular shape changes by affecting the ionic activities of the predominant intracellular cation (K⁺) or anion (Cl^?).     

Fouling deterrent chemical defence in three muricid gastropod egg masses from the Southeast coast of India

Abstract

The egg masses of the marine muricid gastropod molluscs Chicoreus virgineus, Chicoreus ramosus and Rapana rapiformis were studied for antifouling activities. The minimum inhibitory concentrations of crude extracts for the inhibition of byssal production and attachment of the brown mussel Perna indica were 650 μg ml^?1, 1150 μg ml^?1 and 925 μg ml^?1 from the three muricid gastropods, respectively. Higher LC₅₀ values than EC₅₀ values and 100% recovery of the mussels in the toxicity assay indicated the non-toxic nature of the extracts. The gradient partitioning of the egg mass extracts and subsequent antimicrofouling screening against 40 biofilm bacteria showed wide-spectrum antibacterial activity of the medium polar fraction from C. virgineus; the non-polar fraction from R. rapiformis and both non-polar and medium polar fractions from C. ramosus. The antimicrofouling activity from extracts of the three egg masses was found to be more prominent than antimacrofouling activity. This may be attributed to the targeting of a defence strategy against microbes in order to protect the developing mollusc embryos.     

The ultrastructural organization of the isolated cortex in eggs ofNassarius reticulatus (Mollusca)

Summary We have studied the organization of the cortex in fertilized eggs ofNassarius reticulatus by examining rotary-shadowed whole mounts of isolated cortices in the transmission electron microscope. The following components were distinguished: (a) the plasma membrane, with clathrin-coated areas and coated pits, (b) microfilaments and microtubules, and (c) a tubulovesicular network of endoplasmic reticulum. Microfilaments were identified by labeling with heavy meromyosin, and microtubules with a monoclonal anti-tubulin antibody, using both immunofluorescence microscopy and immunogold labeling for transmission electron microscopy. The microfilaments are organized in a network parallel to and closely associated with the plasma membrane, with typical Y- and X-shaped intersections. The endoplasmic reticulum is associated with this microfilamentous lattice. The microtubules also run parallel to the plasma membrane, but they are located at a greater distance, as can be inferred from stereo images. In the uncleaved egg, numerous microtubules are present in the egg cortex. Shortly before polar lobe formation, at the onset of mitosis, the microtubules disappear almost entirely. They reappear again at the end of first cleavage, as the polar lobe is being resorbed. The synthesis of cortical microtubules at this stage appears to depend on the presence of microtubule-organizing centers in the animal hemisphere of the egg, since microtubules do not reappear in isolated polar lobes. Clathrin-coated areas are present in both the animal and vegetal hemisphere before polar lobe formation. During mitosis, the clathrin-coated plaques and pits are found almost exclusively in the animal hemisphere. After resorption of the polar lobe, at the two-cell stage, no clathrin-coated areas were found at all.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta. II. Large bleb formation caused by high concentrations of exogenous calcium ions

Fertilized eggs of the mollusk Ilyanassa obsoleta (Nassarius obsoletus) form large blebs resembling polar lobes within 12 min of exposure to solutions of isotonic CaCl₂, whereas control eggs in sea water remain spherical. Under identical conditions, fertilized eggs of the sea urchin, Strongylocentrotus purpuratus, an organism which normally does not form polar lobes, do not form blebs upon exposure to solutions of isotonic CaCl₂. The calcium-induced blebbing in Ilyanassa still occurs if other cations such as Na⁺, Mg²⁺, or Mn²⁺ are present in addition to Ca²⁺, but not if comparable concentrations of K⁺ are present. Cytochalasin B prevents the calcium-induced blebbing, whereas colchicine does not. Cytokinesis in both Ilyanassa and Strongylocentrotus and normal polar lobe formation in Ilyanassa appear to require exogenous K⁺ but not exogenous Ca²⁺. Preliminary electron microscopy of Ilyanassa eggs exposed to isotonic solutions of CaCl₂ has shown microfilaments in the cortical cytoplasm in the region of the bleb constriction but no microfilaments in spherical control eggs in sea water. These data suggest that high concentrations of exogenous Ca²⁺ trigger the polymerization and contraction of a ring of microfilaments in the cortical cytoplasm of the Ilyanassa egg which results in the formation of a lobelike bleb of cytoplasm. The observation that K⁺ antagonizes this Ca²⁺-induced blebbing has led to the formulation of a theory which postulates that the ratio of intracellular Ca²⁺ to intracellular K⁺ is critical in the control of polar lobe formation and cytokinesis.     

The Hippo pathway controls polar cell fate through Notch signaling during Drosophila oogenesis

During Drosophila oogenesis, the somatic follicle cells form an epithelial layer surrounding the germline cells to form egg chambers. In this process, follicle cell precursors are specified into polar cells, stalk cells, and main-body follicle cells. Proper specification of these three cell types ensures correct egg chamber formation and polarization of the anterior–posterior axis of the germline cells. Multiple signaling cascades coordinate to control the follicle cell fate determination, including Notch, JAK/STAT, and Hedgehog signaling pathways. Here, we show that the Hippo pathway also participates in polar cell specification. Over-activation of yorkie (yki) leads to egg chamber fusion, possibly through attenuation of polar cell specification. Loss-of-function experiments using RNAi knockdown or generation of mutant clones by mitotic recombination demonstrates that reduction of yki expression promotes polar cell formation in a cell-autonomous manner. Consistently, polar cells mutant for hippo (hpo) or warts (wts) are not properly specified, leading to egg chamber fusion. Furthermore, Notch activity is increased in yki mutant cells and reduction of Notch activity suppresses polar cell formation in yki mutant clones. These results demonstrate that yki represses polar cell fate through Notch signaling. Collectively, our data reveal that the Hippo pathway controls polar cell specification. Through repressing Notch activity, Yki serves as a key repressor in specifying polar cells during Drosophila oogenesis.     

Polar effects of concanavalin A on the cortical cytoskeleton of a molluscan egg (Nassarius reticulatus,Gastropoda)

Summary The organization of the surface of fertilizedNassarius reticulatus eggs was probed by investigating the effects of treatment with concanavalin A (Con A). This lectin causes abnormal polar lobe formation as well as inhibition of cleavage. At low concentrations of Con A (0.3–1.0 μg/ml) the polar lobe constriction becomes considerably elongated, whereas at higher concentrations (2.5–50 μ/ml) the position of the constriction undergoes an extreme shift towards the animal pole. In the latter case, the surface of the animal part of the egg forms large protrusions and folds. Con A also causes resorption of microvilli and disappearance of the extracellular layer covering these villi; this process starts at the vegetal pole and propagates towards the animal pole. These changes in surface architecture are associated with profound changes in the organization of filamentous (F-) actin as assessed by confocal laser scanning microscopy of NBD-phallacidin-labelled eggs. Divalent succinyl-Con A has the same effects on polar lobe formation and surface architecture as tetravalent Con A, but only at very high concentrations (100–200 μg/ml), indicating that Con A exerts its effects by cross-linkage of its binding sites. Experiments with cytoskeleton inhibitors (cytochalasin D, colchicine, and nocodazole) reveal that in Con A-treated eggs — as in untreated eggs — microfilaments, but not microtubules, are involved in the formation of the polar lobe constriction. The calcium ion channel blocker D600 affects neither normal nor Con A-induced abnormal polar lobe formation, which suggests that influx of external calcium is not required. In contrast, treatment with TMB-8, an antagonist of internal calcium release, prevents the formation of a polar lobe in both normal and Con A-treated eggs. Finally, eggs from which the polar lobe has been removed prior to Con A treatment show none of the effects described, whereas isolated polar lobes react similarly to intact eggs. These results suggest that binding of Con A to sites present at the vegetal pole of the egg is responsible for the observed effects of the lectin.