Sven Hörstadius

Vegetalization of sea urchin larvae by chloramphenicol

Abstract Chloramphenicol (chloromycetin), an inhibitor of protein synthesis, is shown to bring about a vegetalization of whole sea urchin eggs as well as of animal and vegetal halves of such eggs.

Morphogenetic agents from unfertilized eggs of the sea urchin Paracentrotus lividus

Abstract Previous experience points to the conclusion that specific morphogenetic agents form the basis of differentiation in the early development of sea urchins. Attempts have therefore been made to isolate such agents from the unfertilized eggs of the sea urchin, Paracentrotus lividus . By ion-exchange chromatography on Dowex 50 W-X2 of partly purified egg homogenates a fraction was isolated which showed an animalizing action on whole eggs and embryos. When tested on animal and vegetal halves, isolated in the 16- and 32-cell stages, the animalizing effect was considerably increased. The fraction caused a definite extension of the ciliary tuft in animal halves, and it was capable of inducing a differentiation to almost normal plutei on vegetal halves. A treatment for 2 hours was found sufficient to induce the effect if it was applied during the period of competence, i.e., the first 6 hours after the operation. A second fraction eluted from the column in close proximity to the animalizing one caused a vegetalization which, however, was somewhat weaker, particularly when tested on whole eggs and embryos. When animal halves were exposed to animalizing and vegetalizing fractions mixed in certain proportion, a neutralization of their effects was observed.

Morphogenetic substances from sea urchin eggs. Isolation of animalizing and vegetalizing substances from unfertilized eggs of Paracentrotus lividus

Abstract A method is described for the isolation of morphogenetic substances from mature, unfertilized eggs of the sea urchin Paracentrotus lividus. A complete separation of several different substances with specific animalizing or vegetalizing effect on the early development of sea urchins, when tested on whole eggs and on isolated animal and vegetal halves, was achieved by chromatography on Dowex 50 W-X2 and on Sephadex G-25. The results suggest that developmental processes can be influenced by various types of substances that may preexist in the unfertilized eggs. Two of the animalizing substances were obtained extensively purified. According to their ultraviolet absorption curves, on may contain tryptophan and the other consist of a nucleotide. They both exhibit a very high specific activity but differ significantly in their morphogenetic specificity. One induces strong animalization when tested on animal halves but has no effect when tested on whole eggs or on vegetal halves. In contrast, the other shows a strong animalizing activity in all three test systems. The high specific activity of the purified animalizing substances is demonstrated by their ability in very low concentrations to force the animal halves into their most extreme animal type of differentiation, i.e., with stereocilia all around the blastula wall, and to induce a differentiation on the vegetal halves not only to plutei but to a new extreme animalized larval type, the radial type. This type of vegetal half is characterized by the presence in the early stages of an apical tuft, and a further development with a thick ectoderm covering about half the body and with the skeleton in equatorial and radial position.

Studies on controlled and released respiration in animal and vegetal halves of the embryo of the sea urchin, Paracentrotus lividus

Summary Eggs of the sea urchin Paracentrotus lividus were separated in the 16–32 cell stage in animal and vegetal halves. These were allowed to develop, and the respiration of the halves was measured in the microrespirometer of Linderstrom-Lang and Holter. The measurements referred to advanced cleavage stages (≥128 cells) and to the mesenchyme blastula stage or beginning gastrula stage. The medium was either pure sea water or sea water within the addition of 5×10 −5 M 2,4-dinitrophenol. In the first medium the “controlled” in the latter the “released” respiration was measured. A survey of the results is given in Figs. 1 and 2. In both stages which were subject to examination, the respiratory control ratios (released respiration over controlled respiration) in animal halves were high, about 2.9, a value corresponding to that found immediately after fertilization in whole eggs suspended in normal sea water [14]. In advanced cleavage stage, the respiratory rate seemed to be higher in vegetal than in animal halves. Addition of dinitrophenol caused a certain decrease in the respiratory rate (Fig. 1). Vegetal halves in the mesenchyme blastula state (Fig. 2), on the other hand, showed a respiratory rate not significantly different from that in animal halves. Upon addition of dinitrophenol only a rather low increase in the respiratory rate occurred (Fig. 2). Controls were made in order to ascertain that the changes in respiratory rate were not due to damage caused by the process of separating the halves. As the released respiration expresses the maximal electron transport capacity [24], it may also be considered an approximate measure of the amount of respiratory enzyme chains present in the embryos. Thus, a considerable synthesis of these enzymes must take place in the isolated animal halves. This is in agreement with the increasing incorporation of amino acids in isolated animal halves, demonstrated by Markman [21]. The results with isolated vegetal halves at mesenchyme blastula stage indicate, on the other hand, that the amount of respiratory enzyme chains is lower than in animal halves, which again corresponds to studies on incorporation of labeled amino acids [21]. The different values of the respiratory control ratio in animal and vegetal halves indicate differences in metabolic state. A high value of the ratio is in keeping with an accumulation of energy-rich compounds, whereas lower values indicate that a certain steady state has been approached. The interactions between animal and vegetal regions which display themselves in the normal development are discussed in the light of the results obtained. Financial support was given from the Swedish Natural Sciences Research Council, the Swedish Cancer Society and the Consiglio Nazionale delle Recerche (Rome). These grants are most gratefully acknowledged.

Change of distribution of mitochondria in animal halves of sea urchin eggs by the action of micromeres

Abstract The mitochondrial pattern of an isolated animal egg half agrees with that of a larva animalized by iodosobenzoic acid, while the pattern of an isolated vegetal half is consistent with that of a lithium vegetalized larva. Implantation of micromeres into an animal half shifts the pattern into that of a normal or a vegetalized larva. The strength of the effect is a function of the number of micromeres implanted.

Differentiation of the sea urchin egg following reduction of the interior cytoplasm in relation to the cortex

Abstract 1. 1. The central cytoplasm of eggs of Echinus esculentus L. and Psammechinus miliaris (Gmelin) was reduced by suction before fertilization. The total cytoplasmic reduction amounted to about 50 per cent. 2. 2. In 20 eggs which started development no deviations in either animal or vegetal direction were observed. The resulting plutei were normal in every respect except size. 3. 3. The results are contrary to Dalcqs concept of the differential localization of gradients at the centre and periphery of the sea urchin egg.

A note on the effect of temperature on sea urchin eggs

Abstract Ripe animals of Paracentrotus lividus are found at Naples all the year round, but the temperature range for normal development of winter eggs and of summer eggs is different. The winter and summer eggs also behave differently at extreme temperatures. Animal halves of spring eggs at 13°C become animalized in comparison to halves reared at 23°C. In contrast, vegetal halves become vegetalized. As the changes counteract each other the balance between the animal and vegetal systems will probably not be disturbed in spite of changes in the two metabolic systems.

The incorporation of 35SO4= in whole embryos and meridional, animal and vegetal halves of the sea urchin Paracentrotus lividus

Abstract Measurements are presented (Tables I and II) concerning the incorporation of 35 SO 4 = into whole embryos, into meridional, animal and vegetal halves. The measurements refer to early gastrula stage with inclusion of data pertaining to later stages of gastrulation and to complete gastrulae. When the data are referred to equal volume no significant differences are found between the different categories of embryos (whole embryos or halves of different kinds). This indicates that the rate of uptake of sulfate may be about equal in the different regions of the embryo, and this is not in disagreement with available histochemical and autoradiographic data. These results are in contrast to the strikingly different effects of deprivation of sulfate on animal and vegetal regions in whole embryos and on animal and vegetal halves, as known from previous work [13, 19]. The concentration of available sulfate may not differ, but sulfate may be required to different extent in the different embryonic regions.

Fertilization and membrane formation of sea urchin eggs aspirated in capillaries

Abstract Unfertilized eggs of Psammechinus miliaris were aspirated into capillaries with an inner diameter lesser than that of the egg. As a consequence this assumed an elongated form. The part of the egg which precedes at the aspiration is called the front, the opposite the hind end of the egg. The aspirated egg was fertilized at its hind end. Under these conditions a fertilization membrane appeared at the front end, the membrane formation being inhibited at the hind end and in the part of the egg surface that was in contact with the glass wall of the capillary. In rare cases a membrane was formed also at the hind egg pole. At the contact surface a granular or corrugated membrane may also appear. This membrane adhered to the egg surface. The results prove that the activation impulse propagates from the site of sperm entrance at the hind pole to the front pole, although a large intermediate region is incapable of membrane formation. The phenomenon described is referred to a polarization brought about by the aspiration of the egg, the front end becoming more stretched, the hind end more compressed (Lindahl (4), Dan and Okazaki (2)). The polarization affects probably the vitelline membrane and deeper layers of the cortex as well.

Two metabolic systems with different reactions to temperature in sea urchin larvae

Abstract Animal and vegetal halves of 16-cell stages of Paracentrotus lividus were reared at 23 and 13 °C. Cleavage and differentiation were not hampered within this temperature range. At 13 °C the animal halves became animalized and the vegetal halves vegetalized as compared with the halves reared at 23 °C. This indicates the existence of animal and vegetal metabolic systems with different temperature coefficients. It also suggests an unchanged an/veg balance in whole embryos at different temperatures, since the systems are affected in opposite ways.