J. Immers

The role of mucopolysaccharides in the fertilization of the sea urchin egg

Abstract This paper gives a survey of a number of facts which point to the conclusion that the cortical layer of the unfertilized sea urchin egg contains acid mucopolysaccharides which act as enzyme inhibitors. The natural or artificial activation of the eggs probably causes a break-up of the inhibitory substances and thus releases certain enzyme systems. The results of staining the eggs by the methods of Hotckhiss and Hale seem to bear out these conclusions.

Studies on cytoplasmic components of sea urchin eggs stratified by centrifugation

Abstract The participate components of the sea urchin egg cytoplasm stratified by centrifugation were studied in living and fixed material. The acetic iron, PAS and methyl green-pyronin stainings served as histochemical methods. The unfertilized eggs of Arbacia lixula, Echinocardium cordatum, Echinus esculentus, Paracentrotus lividus and Psammechinus miliaris were used as material. Sulphate esterified mucopolysaccharides are present in the cortex of all sea urchin eggs. The mode of distribution of the sulphate and α-glycol groupings varies according to the sea urchin species. This is in line with the species specific variations in the structure of cortical granules. The staining reactions of the cortical granules are summarized in Table I, p. 509. Sulphate esterified mucopolysaccharides are not only found in the cortex. The interior cytoplasm exhibits, after centrifugation, a layer of fine granules having the staining properties of acid mucopolysaccharides. There is a considerable variation with respect to the location and configuration of this layer in different sea urchin species. In the Arbacia egg the mucopolysaccharides are accumulated and precipitated as globules or, rather, flakes within the layer of fine granules, cf. Fig. 2 (layer c ). In Echinocardium they form a narrow layer of granules of variable size enclosed in a hyaline matrix, cf. Figs. 7 and 9. In centrifuged Echinus eggs they appear as a discontinuous layer mixed with the centripetal region of the layer of yolk granules, cf. Fig. 18. In Psammechinus egg, however, they accumulate in the light layer ( a ) as a uniform precipitate, cf. Fig. 18. Conversely, sections of the Paracentrotus eggs fails to reveal a layer of fine granules with the staining properties of mucopolysaccharides. The sulphate esterified polysaccharide complex does not seem to be bound to mitochondria. One more compound with the staining properties of acid mucopolysaccharides was found to be present in the yolk granules. In view of the chemically demonstrated presence of sialic acid in the yolk granules [24] it was suggested as possible that the spots stained with the acetic iron reagent contains sialic acid as a component. With respect to the relative density of yolk granules and ground cytoplasm of sea urchin eggs investigated two types of eggs are distinguished: (1) the eggs of Arbacia and Echinocardium with yolk granules which are heavy relative to the ground cytoplasm, and therefore accumulate centrifugally; (2) the eggs of Echinus, Paracentrotus and Psammechinus with yolk granules which are lighter relative to the ground cytoplasm. They therefore accumulate in a more centripetal or intermediary region. RNA and its derivatives are present in all layers of the stratified eggs. It was inferred, however, that in different layers of the centrifuged egg RNA may behave differently with respect to the combination with proteins and availability to staining reaction. A certain relation between the centrifugation and morphogenetic axes has been demonstrated in the egg of Echinocardium . The bearing of the histochemical results on the mechanism of formation of the fertilization membrane and on the determination of the dorso-ventral organization of the egg has been briefly discussed.

Chemical and histochemical demonstration of acid esters by acetic iron reagent

Abstract Hales method using the Prussian blue colour is based on the specific reaction of ferric iron with acid esters (sulphuric or phosphoric acid) and also with carboxyl groups of uronic acids and monoaminodicarboxylic acid residues of proteins in a slightly acid environment. Positive reaction is obtained with polyuronides, polysaccharide sulphuric acid esters, polynucleotides, phosphoproteins and phospholipoids. The further development of Hales method is discussed briefly.

Cytochemical studies of fertilization and first mitosis of the sea urchin egg.

Abstract It has been demonstrated that the yolk granules of the sea urchin eggs give a more intense PAS reaction following than prior to fertilization. A hypothetical scheme has been proposed which accounts for the increase of α-glycol groups occurring upon fertilization (cf. scheme I and II, Fig. 13). The Feulgen staining of the pronuclei and of the syncarion has been studied. The results are given in Fig. 3. They confirm that DNA at least in its ordinary form is absent in the female pronucleus and during a certain period in the syncarion as well (cf. stage f, Fig. 3). Some additional data are given concerning the staining reactions of nucleus and chromosomes. The cytoplasm of the spindle poles is intensely PAS-positive (cf. Fig. 5). The maximum reaction occurs in the prophase. Then the intensity decreases gradually (cf. Figs. 7, 9, 10) and finally the reaction disappears in the telophase. The nuclear substances give a very weak PAS reaction in prophase. In meta- and early anaphase the chromosomes are PAS-positive; conversely they are PAS-negative in late ana- and in telophase. The Hale reaction is occasionally faintly positive in prophase but always distinctly positive in meta- and anaphase (cf. Figs. 10 and 11). The telophase chromosomes are Hale-negative.

Nuclear changes in the course of development of the sea urchin studied by means of Hale staining

Abstract The nuclear changes occurring in the course of the early development of Paracentrotus lividus have been studied by means of the ferric acetate staining method of Hale. If the sections of Carnoy fixed eggs or embryos are subjected to the Hale procedure, very few interphase nuclei become stained. Treatment of the sections with a buffer or a buffered trypsin solution prior to the Hale procedure may induce a staining of the interphase nuclei, which varies according to pH of the buffer, stage of development and position in the embryo, in a way, shown in Text-Figs 1–3. During the early development, the interphase nuclei remain unstained even after pretreatment. At an early blastula stage (6 hr after fertilization), the interphase nuclei become Hale positive, following a pretreatment with 0.0025 per cent trypsin (Figs 1c, d). Only at the stage of free swimming blastulae (14 hr after fertilization), pretreatment with acetate buffer gives a certain staining of the interphase nuclei, a staining that then increases in intensity and becomes less pH dependent during the following stages of development (Figs 2 e-j). The prophase chromosomes are not directly stained by the Hale reagent, but staining may be induced in any stage of development by pretreatment with buffer at pH above 3, or with trypsin within the pH range of activity of this enzyme. At pH values above 3, the meta- and anaphase chromosomes become stained without any pretreatment of the sections. At early blastula stage, the Hale staining of the interphase nuclei is not influenced by exposure to DNase or RNase. If, however, the sections are pretreated with trypsin and thereafter exposed to a mixture of DNase and RNase, the staining capacity of the interphase nuclei is abolished. In stages from the late blastula stage onwards, DNase decreases or abolishes the Hale reaction without any previous pretreatment. RNase may affect, but does not alone abolish the staining. The inference from the data is that Fe 3+ in the Hale reagent competes with nuclear proteins for reactive sites in the nucleic acids. These sites may correspond mainly to the phosphate groups. In the interphase nuclei, the proteins block the access of Fe 3+ to the phosphate groups with a strength varying according to the circumstances defined above. In prophase nuclei, the effect of the proteins is weaker and the Hale staining is therefore easily provoked upon pretreatment. In the meta- and anaphase chromosomes, Fe 3+ eliminates without pretreatment the action of proteins. The Hale staining of interphase nuclei is different in different embryonic regions and it often seems to reflect different intensities in synthetic activity. The propensity of interphase nuclei for Hale staining seems to parallel their nucleolar activity and consequently their production of ribosomal RNA. It may also parallel certain changes in pattern of messenger RNA formation during development.

Metabolism of glucosamine in the early sea urchin development.

Abstract By aid of chromatographic assay the glucosamine content has been determined in different stages of development of the sea urchin Paracentrotus lividus. Some data are also given for eggs of Arbacia lixula and Echinus esculentus. There is a general trend towards a decrease of the glucosamine content per egg or embryo during the development. It is about 24 per cent lower in the pluteus larva as compared with the unfertilized egg and 46 per cent lower in the pluteus stage if it is compared with the fertilized egg in the syncarion stage, cf. Fig. 3. The decrease is, however, not a steady one but the values fluctuate showing certain pronounced maxima and minima, as illustrated in Fig. 3. The fluctuations compare very well with those demonstrated by Kavanau for total protein in Paracentrotus embryos. A decrease in the glucosamine content coincides with an increase in total embryonic protein and an increase with a breakdown of yolk proteins. It is therefore tentatively suggested that glucosamine plays a role in protein synthesis. It might act as an amine donor or acceptor. Reference is made to the work of Fulton and co-workers which points to the role of peptide amides in protein synthesis. The glucosamine estimated in this study forms probably part of mucopoly-saccharide molecules. The substance determined as glucosamine may—before hydrolysis—partly represent a combination of glucose residues and unsubstituted amino groups. Its possible role as a component of sialic acid is emphasized.

Treatment with lithium as a tool for the study of animal-vegetal interactions in sea urchin embryos

SummaryThe question about the nature of the effect of lithium on early sea urchin development is reexamined. Essential features of the morphogenetic changes of lithium treated embryos are followed with continuous comparisons with control embryos. The key to the lithium effect is the greater cytoplasmic susceptibility in the animal polar region as compared with the vegetal one. This is diagrammed in Fig. 23a-c on the basis of the double gradient concept. There is a decline of the animal gradient with increasing lithium concentration. As a consequence the level of differentiation of the terminal region becomes more and more vegetal, seean/veg-values in Fig. 23. The region suppressed by a prolonged exposure to lithium, e.g. 9–16 hours, cannot be restored. Nevertheless, there are data from previous research supporting the view that the primary effects of lithium are reversible within certain limits. However, when the normal balance is disturbed by decline of the animal gradient and particularly by suppression of its higher levels, there is a compensatory enhancement of the vegetal gradient system which stabilizes the suppression. As a consequence of the suppression of the higher animal levels, a certain accumulation of cells in an anterior direction has taken place in the blastula stage. The degree of accumulation reflects the degree of vegetalization. Later there is to varying extent a backflow of cells in the vegetal direction. It was shown how a great part of the blastula wall may have the aspect of an attachment zone (Fig. 7). The primary mesenchyme cells attach themselves only to a certain level of the ectoderm in which the relativean/veg-value is around 0.7 according to the conventions behind Fig. 23.Sections of Carnoy fixed embryos were exposed to trypsin. It proved that the external cytoplasm of blastodermic cells in lithium treated embryos was more strongly attacked than the internal one. The latter showed a strong resistance to tryptic action. On the other hand, in the control embryos the inner part of blastodermic cells was completely digested with exception of the vegetal region including the attachment zone. The trypsin resistant structure may be preformed and may be responsible for the higher rigidity of the cytoplasm in lithium treated embryos (section IIIf).It is proposed that in the period of lithium susceptibility, the colloidal state is most affected in the animal region, thereby creating a block to the diffusion of the animalizing substances which results in the shifts diagrammed in Fig. 23.

Heteromorphic budding in lithium-treated sea urchin embryos: A study of gene expression

Abstract After treating eggs and embryos of the sea urchin Paracentrotus lividus with low doses of lithium (e.g. 32.5 mM for 8–10 h) and ensuing rearing in normal sea water, organized protrusions or “buds” may appear ca 40 h after fertilization. Their site of formation is especially the animal region of the entoderm which is enlarged by lithium treatment. They may also appear, although at lower frequency, in the vegetal region of the entoderm. After combined treatment with lithium and trypsin the formation of buds occurs at the most vegetal region of the embryo. The ectodermic nature of the bud epithelium is ascertained on the basis of several criteria. In the primary ectoderm of lithium-treated embryos, the most animal region is suppressed and reduced. In this and in other respects, the buds appear as reduced copies of the primary ectoderm. Certain groups of genes are expressed in embryonic regions in which they are not expressed normally. This is interpreted as being due to a decline in the main control systems of the embryo, the gradients of diffusing animalizing ( an ) and vegetalizing ( veg ) agents. Experiments with actinomycin D and with combined exposure to lithium and trypsin give further support to this interpretation.

Changes within the chromatin during sea urchin embryogenesis: A study of ectodermic interphase nuclei with three-valent acetic iron (Hale) reagent

Abstract The three-valent acetic iron (Hale) staining method is re-examined. The Hale stainability of the interphase nucleus can be increased by pretreatment of the material with buffer solutions of appropriate pH-values, respectively with trypsin. On the basis of staining results three different nuclear reaction periods of embryogenesis can be defined: period I (0–7 h after fertilization), II (7–12 h) and III (12–24 h, cf fig. 1). It is proposed that the variation in nuclear stainability depends on interactions between DNA and histones, the composition of which gradually changes as-development proceeds. It is suggested that the nuclear stainability may be used as a semi-quantitative measure of the readiness of the chromatin tin to participate in transcriptional activity.