Effect of antimediator preparations on the intercellular relations in early embryos of the sea urchin]

The dated treatment of the early embryos of an irregular (flat) sea urchin Scaphechinus mirabilis by neuropharmacological drugs (anti-neurotransmitters) during one of the first four cleavage divisions results in the impairment of intercellular connections and leads to the formation of twin embryos, dwarf embryos, embryos of the dumb-bell shape etc. In the experiments with some of the drugs under study such developmental abnormalities were not seen or were expressed much more weakly when serotonin or bufotenin (N,N-dimethylserotonin) were added to the medium. A suggestion is put forward that the early embryos possess an intracellular mechanism participating in the interaction between the cells and operating via endogenous monoamines, primarily serotonin.     

Sensitivity of fragmented and stratified sea urchin embryos to cytotoxic neuropharmacological preparations

The red half embryos and related quarter embryos (yolk and pigment) of Arbacia lixula, obtained by means of centrifugation of the eggs in sucrose gradient, retain the normal level of sensitivity and supersensitivity to cytotoxic neuropharmaca, antagonists of biogenic monoamines. The white half embryos and clear quarter embryos practically lack supersensitivity whereas the granular quarter embryos restore it to the initial level. The non-pigmented blastomers of stratified embryos are characterized by somewhat weakened supersensitivity. A suggestion is put forward that the supersensitive embryos of A. lixula possess a sensibilizing factor which couples the supersensitivity receptors with the processes of cell division and moves together with the yolk granules upon centrifugation. This factor is not observed in the Strongylocentrotus granularis embryos lacking evident supersensitivity.     

Reception and extrareceptor binding of cytostatic serotonin antagonists by early embryos of the sea urchin Arbacia lixula

Unfertilized eggs and early embryos of the sea urchin Arbacia lixula incubated for 60 min in a medium containing the antagonists of prenervous serotonin, i.e. inmecarb (21 microM) or imipramine (40 microM), bind up to 5 microM of these drugs per 1 ml of cells. At high cell concentrations (more than 10,000 eggs or embryos per 1 ml), this binding is not followed by inhibition of cleavage divisions or by increase in the sensitivity to cytostatic effects of these drugs, which is taken as an indication that this binding is a nonreceptive one. The decrease in concentration of eggs or embryos does not affect total binding of the drugs, although their antiserotonin effects become evident indicating the existence of the receptor sites of binding. In experiments with 3H-imipramine, two binding pools were found (Bmax being correspondingly equal to about 20 and 0.75 microM/ml of embryos; the values of Kd amount to 200 and 15 microM). One of them is a nonreceptive pool, whereas the other presumably coincides with receptor binding sites of prenervous serotonin antagonists.     

The contractility of elongated microvilli in early sea urchin embryos

Summary Elongated microvilli attach the early sea urchin embryo to the fertilization envelope and support it in a concentric position within the perivitelline space. The contractility of the elongated microvilli was demonstrated in several ways. (1) During normal cleavage, these microvilli change their length to adapt to the change in shape and numbers of blastomeres. (2) When treated with calcium-free sea water, embryos become eccentrically located and the microvilli extend further than normal on one side; when returned to normal sea water, the embryos become centered again. (3) Several agents cause the fertilization envelope to become higher and thinner than normal and the elongated microvilli to extend correspondingly if treated within ten min after fertilization. In some cases, both elongated microvilli and fertilization envelope return to normal size when returned to normal sea water. (4) Fertilization in a papain solution causes the elongated microvilli and the fertilization envelope to contract to the surface of the embryo. (5) Refertilization after the papain-induced contraction can bring about the elongation of these microvilli and the elevation of the fertilization envelope a second time. It was also shown that elongated microvilli are extended immediately upon fertilization, at the same time as the short microvilli. The firm adherence of the tips of elongated microvilli to the fertilization envelope by means of extracellular matrix fibers is shown in a high voltage electron microscope stereoimage. This allows us to understand why it is that when the elongated microvilli extend or contract, the fertilization envelope also extends and contracts accordingly.     

Similarity of proteins synthesized by isolated blastomeres of early sea urchin embryos

The 16-cell sea urchin embryo has blastomeres of three distinct size classes: micromeres, mesomeres, and macromeres. Each class is already restricted in its developmental fate, micromeres being committed to formation of primary mesenchyme cells. The three classes of blastomeres were isolated in high purity and incubated in [³⁵S]methionine until the next cleavage. Nearly all the radioactive protein was solubilized and subjected to two-dimensional electrophoresis according to O'Farrell. Of approximately 1000 spots resolved, there are no qualitative differences among the three blastomeres. When embryos were labeled between the first and fourth cleavages and blastomeres then isolated, no qualitative differences in protein synthesis were observed. Moreover, there are very few changes when unfertilized eggs are compared to 16-cell embryos. Thus cellular determination during embryonic development is not accompanied by qualitative changes in the distribution within the embryo of abundantly synthesized proteins, virtually all of which are coded for by sequences present in the egg.     

Cholinergic receptors in early (pre-nervous) sea urchin embryos

Agonists of nicotinic acetylcholine receptors (nAChR) nicotine and 1-acetyl-4-methylpiperazine do not act on the early sea urchin embryogenesis but evoke calcium shock in both oocytes and early embryos under certain conditions. Many nAChR ligands protect both oocytes and embryos against this shock. There seem to exist putative nAChR on the cell surface of the early sea urchin oocytes and early embryos. Pre-nervous acetylcholine seems to be functionally coupled via these receptors with the second messengers, endogenous activators of the protein kinase C.     

Effect of calcium ionophore A23187 on the sensitivity of early sea urchin embryos to cytotoxic neuropharmacological drugs

The ability of cytotoxic neurochemicals (indole and amphetamine derivatives) to block first cleavage division in the embryos of the sea urchin Arbacia lixula abruptly increases when the embryos are incubated in calcium-free seawater and decreases when the external Ca concentration is raised up to 46.4 mM. Sensitivity of the embryos to these drugs decreases also in the presence of the Ca-ionophore A23187. It is suggested that Ca ions are involved in the realization of physiological effects of "prenervous" neurotransmitters whose presence in early sea urchin embryos was shown by us earlier.     

Possibility of membrane reception of neurotransmitter in sea urchin early embryos

1. Methiodide derivatives of serotonin blockers—inmecarb and K.Yur-14 which poorly penetrate the cells specifically affect the pattern of cleavage division in half-embryos of Paracentrotus lividus and Scaphechinus mirabilis.2. Specific [³H]8-OH-DPAT binding under conditions strictly limiting penetration of the ligand into the cells of Strongylocentrotus intermedius was also shown (K_d ∼ 3 × 10⁻¹⁰M for a site with the greatest affinity).3. On the basis of the data obtained it is concluded that neurotransmitter-specific membrane receptors may be present in sea urchin embryos during cleavage divisions. The “protosynapse” hypothesis is proposed which suggests the existence of a specific structure responsible for early blastomere interaction involving transmitters.     

Thymidine uptake by developing sea urchin embryos

Unfertilized Paracentrotus lividus eggs accumulate very little thymidine. Upon fertilization, however, uptake increases sharply. The pool for thymidine and/or its metabolic products is saturated after 40 min of exposure. Its size is expandable and proportional to the initial concentration of thymidine in the medium. The uptake rate is low shortly after fertilization, increases until 40 min after fertilization and remains constant thereafter. Of the radioactivity taken up in the form of [³H]thymidine during a 30 min exposure beginning at 60 min after fertilization, about 1% is associated with the acid-insoluble fraction and 99% with the acid-soluble fraction.     

Serotoninergic processes in cells of early embryos of the sea urchin Paracentrotus lividus

Agonists of serotonin receptors generate specific inward currents in the cells of early embryos of sea urchin Paracentrotus lividus. Dose-dependent current generated by 5-HT3-agonist 5-HTQ shows complex volt-amperic characteristics with reversal potential -25 and +15 mV. The 5-HTQ effect seems to be due to the activity of channels of mixed conductivity. The 5-HTQ effect is more obvious during cleavage furrow formation. The findings suggest presence of serotonin receptors in the surface membrane of blastomers and their activity play a certain role in regulation of cellular events during cleavage division in the early sea urchin embryo.