Activation of ascidian eggs with lectins

Eggs of Phallusia mammillata, dechorionated with trypsin, were activated in solutions of concanavalin A and wheat germ agglutinin. The polar bodies were extruded, and an aster appeared, but there was no cleavage. Ooplasmic segregation took place and mitochondria accumulated at the vegetal pole. Large chromatic granules formed in older eggs. The striking parallel between the migration of lectin receptors to the vegetal pole and “capping” was noted.     

Experimental activation of ascidian eggs.

The effects of the ionophore A23187 on the activation of the eggs of Ascidia malaca have been studied. No common external ion in the sea water is found to be essential for the activation but lanthanum and manganese inhibit the response. These observations support the interpretation that activation of these eggs results from changes in free intracellular calcium levels. This has led to the prediction of two other activating treatments, namely high external calcium and addition of theophylline.     

Periodic calcium waves cross ascidian eggs after fertilization

Ascidian eggs respond to fertilization with one to two dozen periodic calcium pulses (J.E. Speksnijder, D.W. Corson, C. Sardet, and L.F. Jaffe, 1989a, Dev. Biol. 135, 182-190). We examined the spatial pattern of these pulses and found that they are initiated in discrete regions from which they propagate as waves. The first few pulses start in the animal hemisphere, whereas the later ones are mostly initiated near the vegetal pole. Such vegetal waves are often followed by a contraction of the egg surface. Since these waves are attenuated as they spread, they repeatedly expose the vegetal pole region to more calcium. The mechanism of these repetitive calcium waves and their possible role in establishing pattern or completing meiosis is discussed.     

Evidence for cell surface and internal phospholipase activity in ascidian eggs

Upon fertilization, ascidian eggs release a cell surface glycosidase used in the block to polyspermy and undergo cortical contractions resulting from increased intracellular calcium levels. The glycosidase is released by fertilization, calcium ionophores or added phospholipase C (PLC) activity. The PLC inhibitor D609 blocks glycosidase release. Intact Ascidia ceratodes eggs cleave 4-methylumbelliferyl-phospho-choline when it is added to seawater. This yields highly fluorescent 4-methylumbelliferone. Authentic phospholipase C but not phospholipase D can cleave this substrate. Thus, the authors believe that cleavage of the substrate is specific for PLC activity. Eggs incubated in the fluorogenic substrate after having been washed and detergent extracted were not fluorescent. Therefore the substrate failed to enter intact cells. Glycosidase release and PLC activity were stimulated by ionomycin. Octylglucoside or Triton X-100 extracts of ascidian eggs had two forms of phospholipase activity as shown by ion affinity chromatography: PL1 eluting at 0.25 mol/L NaCl and PL2 eluting at 0.6mol/L NaCl. The PL1 appeared to be isolated as a single protein. When surface proteins were labeled with non-penetrating biotin and were subsequently reacted with streptavidin, half of the PLC activity bound. This demonstrates that half the ascidian egg PLC activity is located on the surface of either the egg or follicle cell, and half is located within the egg.     

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.     

A new method for evaluation of cavitation near mechanical heart valves

Evaluation of cavitation in vivo is often based on recordings of high-pass filtered random high-frequency pressure fluctuations. We hypothesized that cavitation signal components are more appropriately assessed by a new method for extraction of random signal components of the pressure signals. We investigated three different valve types and found a high correlation between the two methods (r2: 0.8806-0.9887). The new method showed that the cavitation signal could be extracted without a priori knowledge needed for setting the high-pass filter cut off frequency, nor did it introduce bandwidth limitation of the cavitation signal.     

L-type Ca2+ currents in ascidian eggs.

We have studied Ca2+ currents in ascidian eggs using the whole-cell clamp technique. T and L components, as observed in somatic cells, are present and the L-type current predominates. Since the IV relationship for these inward currents overlap at -30 mV, separation of the two components using different voltage regimes is not feasible. Increasing external Ca2+ results in larger currents. The L-type current decreases in a dose-dependent fashion in the presence of Mn2+ and Nifedipine, while the T-type current is inhibited in Ni2+. When Ba2+ was used as the carrier ion, channel kinetics and conductance were completely altered. Considering the density and kinetics of L-type channels in unfertilized eggs it is probable they play an important role in regulating cytosolic Ca2+ during early developmental processes.     

A two-step method for permeabilization of Drosophila eggs

As a first step in developing a procedure for the cryopreservation of Drosophila melanogaster embryos, we have established a method for permeabilization of the eggcase and have initiated studies of the hydraulic conductivity of permeabilized embryos and the permeation of selected cryoprotective agents. The eggcase of D. melanogaster embryos has a wax layer that precludes any flux of water. A two-step procedure employing organic solvents was developed to effect removal of the wax layer with minimal deleterious effects on the embryos. Dechorionated embryos (Oregon-R strain P2, 12 to 13 hr old) were rinsed sequentially in isopropanol and hexane. After removal of solvent, embryos were held in a modified cell culture medium for further manipulation. This procedure routinely yielded 80 to 95% of the eggs permeabilized (as determined by osmotic contraction in 1 M sucrose) and 75 to 90% survival (incidence of hatching). Hydraulic conductivity of permeabilized embryos and permeation of cryoprotectants were determined using a microdiffusion chamber and computerized video microscopy. Regression analysis of the volumetric data from individual embryos yielded the Boyle-van't Hoff function FVeq = 0.124 (osm-1) + 0.541 with the standard deviations of slope and intercept (Vb) being 0.010 and 0.040, respectively. Permeabilized embryos exhibited ideal osmotic behavior over the range of 0.265 to 2.00 osm. The mean hydraulic conductivity coefficient (Lp) was 0.722 +/- 0.366 micron/(min.atm) at 20 degrees C, based on observations of contraction following a step change in concentration of Ringer's solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary and secondary messengers in the activation of ascidian eggs

Two early events of activation in the ascidian egg, the surface contraction and the fertilization current, were studied. Ca ionophore induces contraction without generating a fertilization current, whereas microinjection of IP3 or soluble fractions of homogenized spermatozoa trigger both a contraction and a current. This suggests that the primary trigger of activation in ascidian eggs is a soluble component of spermatozoa that may be released into the egg subsequent to gamete fusion. IP3, or other intermediates in phosphoinositide metabolism, is a putative second messenger that activates fertilization channels directly (probably a Ca-independent process), and subsequently induces surface contraction by releasing Ca from intracellular stores.     

A new method for transduction of mesenchymal stem cells using mechanical agitation

Applications of bone marrow-derived mesenchymal stem cells in gene therapy have been hampered by the low efficiency of gene transfer to these cells. In current transduction protocols, retrovirus particles with foreign genes make only limited contact with their target cells by passive diffusion and have short life spans, thereby limiting the chances of viral infection. We theorized that mechanically agitating the virus-containing cell suspensions would increase the movement of viruses and target cells, resulting in increase of contact between them. Application of our mechanical agitation for transduction process has increased the absorption of retrovirus particles more than five times compared to the previous static method without changing cell growth rate and viability. The addition of a mechanical agitation step increased transduction efficiency to 42%, higher than that of any other previously-known static transduction protocol.     

Fertilization channels in ascidian eggs are not activated by Ca

Using the whole-cell voltage clamp technique, experiments were carried out on ascidian eggs to determine the role of intracellular Ca in the gating of fertilization channels. Raising the level of Ca by adding Ca to the intracellular perfusion medium or by loading the egg cortex (greater than 50 microM) with Ca through voltage gated channels did not lead to the activation of fertilization channels. Alternatively, eggs exposed to low-Ca seawater, perfused with the chelator K-EGTA or Ca channel blocking agents to prevent the release of Ca from intracellular organelles, and subsequently inseminated generated fertilization currents. This argues against Ca as a second messenger in the activation of fertilization channels in the ascidian egg and alternative mechanisms are discussed.     

Interactions between cytoskeletal components during myoplasm rearrangement in ascidian eggs

Ooplasmic segregation in ascidian eggs consists of two phases of cytoplasmic movement, the first phase is mediated by the microfilament system and the second is mediated by the microtubule system. Recently, two novel proteins, p58 and myoplasmin-C1, which are localized to the myoplasm, were suggested to have important roles in muscle differentiation. In order to analyze the molecular mechanisms underlying ooplasmic segregation, the interactions between actin, tubulin, p58 and myoplasmin-C1 were examined. During the first segregation, microtubule meshwork in the unfertilized egg disappeared. At the second segregation, a novel structure of the microtubules that extended from the sperm aster and localized in the cortical region of the myoplasm was found. Moreover, uniform distribution of the cortical actin filament was observed at the second segregation. During the course of myoplasm rearrangement, p58 and myoplasmin-C1 are colocalized and can form a molecular complex in vitro. This complex of p58 and myoplasmin-C1 is a good candidate for a cytoskeletal component of the myoplasm, and is likely to be involved in the correct distribution of cytoplasmic determinants.     

A yellow crescent cytoskeletal domain in ascidian eggs and its role in early development

In this investigation, Triton X-100 extraction was utilized to examine the cytoskeleton of ascidian eggs and embryos. The cytoskeleton contained little carbohydrate or lipid and only about 20–25% of the total cellular protein and RNA. It was enriched in polypeptides of molecular weight (M_r) 54, 48, and 43 × 10³. The 43 × 10³M_r polypeptide was identified as actin based on its M_r, isoeletric point, and affinity for DNase I. Electron microscopy of the detergent-extracted eggs showed that they contained cytoskeletal domains corresponding to colored cytoplasmic regions of specific morphogenetic fate in the living egg. A yellow crescent cytoskeletal domain in the myoplasm was examined and shown to consist of a plasma membrane lamina (PML) and a deeper lattice of filaments which appeared to connect the yellow crescent pigment granules to the PML. The PML probably consists of integral membrane proteins stabilized by an underlying network of actin filaments since NBD-phallacidin stained this area of the egg cortex and the PML was extracted from the cytoskeleton by DNase I treatment. The yellow crescent cytoskeletal domain was found throughout the cortex of the unfertilized egg. During ooplasmic segregation it progressively receded into the vegetal hemisphere and was subsequently partitioned to the presumptive muscle and mesenchyme cells of the 32-cell embryo. It is suggested that contraction of the actin network in the yellow crescent cytoskeletal domain is the motive force for ooplasmic segregation. This structure may also serve as a framework for the positioning of morphogenetic determinants involved in muscle cell development.     

Ooplasmic segregation of the myoplasmic actin network in stratified ascidian eggs

Summary Ooplasmic segregation in ascidians includes the movement of the myoplasm, a pigmented cytoplasmic region thought to be involved in the determination of the embryonic muscle and mesenchyme cell lineages, into the vegetal hemisphere of the egg. A myoplasmic cytoskeletal domain (MCD), composed of a cortical actin network (the PML) and an underlying filamentous lattice extending deep into the cytoplasm, is present in this region. The MCD gradually recedes into the vegetal hemisphere during ooplasmic segregation. It has been proposed that the segregation of the myoplasm is mediated by the contraction of the PML. To test this possibility we have examined ooplasmic segregation in eggs in which the internal parts of the MCD were separated from the PML by centrifugal force. Transmission and scanning electron microscopy of eggs extracted with Triton X-100 showed that the PML remained intact when the internal portions of the MCD were displaced and stratified by centrifugation. When stratified eggs were fertilized there were no rearrangements of the visible cytoplasmic inclusions, but the cellular deformations and the recession of the PML characteristic of ooplasmic segregation occurred as usual. The results indicate that the recession of the PML occurs independently of the internal constituents of the MCD and suggest that PML contraction is the motive force for ooplasmic segregation.     

Spatial distribution of messenger RNA in the cytoskeletal framework of ascidian eggs

Maternal poly(A)⁺RNA, histone mRNA, and actin mRNA exhibit unique spatial distributions in the different ooplasmic regions of ascidian eggs. These RNAs also appear to migrate with their respective ooplasms during the episode of extensive cytoplasmic rearrangement that occurs after fertilization, suggesting they are associated with a structural framework. The role of the cytoskeletal framework (CF) in determining the spatial distribution of maternal mRNA was tested by subjecting Triton X-100 extracted (Styela plicata) eggs and early embryos to in situ hybridization with poly(U) and cloned DNA probes. Grain counts indicated that substantial proportions of the egg poly(A)⁺RNA, histone mRNA, and actin mRNA were present in the CF and that there was no alteration in the extent of mRNA-CF interactions during the period between fertilization and the two-cell stage. Analysis of grain distributions indicated that poly(A)⁺RNA, histone mRNA, and actin mRNA were concentrated in the same regions of detergent-extracted eggs as they are in intact eggs. The proportions and spatial distribution of these RNAs in the CF were not affected when the actin cytoskeleton was destabilized by cytochalasin B or DNAse I. The data suggest that maternal mRNA is associated with the CF, that this association is responsible for mRNA rearrangement during ooplasmic segregation, and that mRNA-CF interactions are not dependent on the integrity of the actin cytoskeleton.     

Effect of Colchicine and cytochalasin B on ooplasmic segregation of ascidian eggs

Summary Ooplasmic segregation inPhallusia mammillata was completed 3 to 5 min after fertilization. Colchicine, which completely stopped nuclear divisions, did not inhibit segregation. Cytochalasin B, which prevented cleavage at a low concentration (0.2 g/ml) inhibited segregation only at a concentration at least five times higher. The action of these drugs leads to the conclusion that ooplasmic segregation does not depend on an assembly of microtubules or on microfilaments which are involved in cell division.This work was performed at the Station Zoologique Villefranche-sur mer (Director: Prof. P. Bougis). The work was supported by a grant A.T.P. of C.N.R.S.     

Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi

 Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis) blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively. Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole, whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole. Recieved: 10 June 1996 / Accepted: 12 September 1996