John Runnström

Atmungsmechanismus und Entwicklungserregung bei dem Seeigelei

VII. VIII. IX. X. XI. XII. XIII. I n h a l t Fragestellung . . . . . . . . . . . . . . . . . . . . . . 106 ~[ethoden . . . . . . . . . . . . . . . . . . . . . . . 109 Uber die Kohlenoxydhemmung der Atmung unbefrnchteter nnd befruchteter Eier . . . . . . . . . . . . . . . . . . . . . . . 110 Theoretisehes fiber die Kohlenoxydversuche . . . . . . . . . . . 116 Uber die ]~emmung der Atmung unbefruehteter und befrnehteter Eier dutch KCN . . . . . . . . . . . . . . . . . . . . . . . 122 Uber die 0xydation des Dimethylparaphenylendi~mins dutch unbefruehtete und befruchtete Eier . . . . . . . . . . . . . . . . . . . 129 Uber die Beeinflnssnng der Atmung dutch Methylenblau . . . . . . . 138 Uber die Beeinflussung der Atmung dureh Rypertonie nnd Narkotika . 146 ]Jber die Beeinflussung der Atmung dureh tIypotonie des Mediums . 150 Atmung~ Struktur und Zellteilung . . . . . . . . . . . . . . . 153 Riiekbliek und Diskassion . . . . . . . . . . . . . . . . . . 163 Zusammenfassung des Inhalts tier verschiedenen Abschnitte . . . . . . 168 Verzeichnis der angeffihrten Literatur . . . . . . . . . . . . . 170

Studies on the surface layers and the formation of the fertilization membrane in sea urchin eggs

Abstract Studies were carried out on the surface layers and the formation of the fertilization membrane in the eggs of Psammechinus miliaris, Echinocardium cordatum and Strongylocentrotus droebachiensis. The sign of birefringence and the retardation of the cortical layer were determined. No perceptible change of retardation occurred on fertilization. The birefringence of the cortex is due chiefly to ordered lipoid layers. The cortical granules have no influence on the birefringence of the cortex. They are formed at the maturation of the egg. By means of a number of different experiments and observations, it was rendered probable that the cortical granules, along with a matrix, merge with the vitelline membrane to form the fertilization membrane. A granular membrane gives a low retardation. Only on incorporation of the cortical granules and their matrix did the retardation of the membrane attain its normal value (about 12.5 mμ). An attempt was made to combine numerous data to a consistent picture of the mechanism of the membrane formation, cf. above under “Discussion.”

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.

Release of respiratory control by 2,4-dinitrophenol in different stages of sea urchin development.

Abstract The aim of this research was to study the respiratory control in different stages of early development until the pluteus stage of the sea urchins Paracentrotus lividus and Psammechinus miliaris . It was started from the well-established fact that certain low concentrations of 2,4-dinitrophenol (DNP) release respiratory control to maximal extent. The respiratory rate in the presence of DNP gives thus the capacity of the respiratory system of the cells examined (cf. upper curves in Figs. 2 and 3). The respiratory control ratio (the DNP-released over the normal respiratory rate) was calculated in a greater variety of material (Figs. 4 and 5, Table 2). The ratio is high in the early postfertilization stage but decreases rather steeply during the first developmental period (from fertilization to the onset of the mesenchyme blastula stage). During the second period (from the mesenchyme blastula to the pluteus stage) the ratio remains almost constant or shows a slope much less steep than in the previous period. The exponential rise of respiratory rate occurring in the first developmental period means a partial release of respiratory control. In view of current experience from studies on mitochondrial systems it is postulated that the partial release results from an increased formation of phosphate acceptors due to the utilization of energy-rich phosphate for syntheses of embryonic components. The respiratory rate prevailing in an early postfertilization stage of Paracentrotus eggs was lower in the winter and early spring than in the late spring. Nevertheless the respiratory control ratio showed no significant variation when the two periods were compared. The respiratory changes occurring upon fertilization are briefly discussed.

The cortical propagation of the activation impulse in the sea urchin egg

Abstract On the basis of observations carried out on eggs attached to a glass surface, it was inferred that the impulse caused by the sperm attachment primarily spreads in the cortical layer. The primary action of the spermatozoon is considered to be propagated by a chain reaction, in which activateable units present in the cortex are concerned. The factual observations are summarized in points 1–5 under “Discussion”, see p. 424.

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.

Die Beeinflussung der Atmung und Spaltung im Seeigelei Durch Dimethylparaphenylendiamin und Hydrochinon

l n h a l t I. Allgemeiner Tei] . . . . . . . . . . . . . . . . . . . . . . . . . . . 532 II . Experimenteller Tei] . . . . . . . . . . . . . . . . . . . . . . . . . 540 a) Methode, Bezeichnungen . . . . . . . . . . . . . . . . . . . . . . 540 b) Der Sauerstoffverbrauch bei versehiedenen Konzentr~tionen yon Dimethylp~raphenylendiamin . . . . . . . . . . . . . . . . . . . . . . . . 541 c) Der respiratorische Quotient bei normalen befruchteten Eiern . . . . . 543 d) Der respiratorisehe Quotient bei Eiern in Dimethylpar~phenylendiamin 546 e) Die Bereehnung a6rob gebildeter fixer S~,ure bei gleichzeitiger Bestimmung des respiratorischen Quotienten . . . . . . . . . . . . . . . . . . . 548 f) Die a6robe Bildung fixer S~ure bei unbefruchteten Eiern in Dimethylparaphenylendiamin . . . . . . . . . . . . . . . . . . . . . . . . . . 552 g) Die Oxydationsvorg~nge im Ei in Gegenwart von Hydrochinon . . . . . 554 h) Die Oxydationsvorgange in gew~rmten Eiern . . . . . . . . . . . . . 558 i) Die a6robe und ana6robe S~ur6bildung in gew~,rmten Eiern ~ . . . . . 559 I I ] . Zusammenfassung der Ergebnisse . . . . . . . . . . . . . . . . . . . 563 IV. Liter~tur . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564

Sperm-induced protrusions in sea urchin oocytes: A study of phase separation and mixing in living cytoplasm

Abstract The phenomena described by Albertsson (1960) as occurring in phase systems are regarded as useful models for the understanding of biological structure. A survey is given of results in respect to phase separation and partition which are observed in fertilized sea urchin eggs after pretreatment with low doses of trypsin. Upon insemination of sea urchin oocytes, the spermatozoa induce the formation of protrusions (reception cones). These consist of ground cytoplasm and form a phase I strictly separated from a more proximal phase II, rich in dispersed endoplasmic vesicles. The rest of the cytoplasm is rich in granular components and may be regarded as a phase III. This view is substantiated by observations on the remixing of components that occurs during the withdrawal of the protrusion. This is a process which is driven by the tendency to remix and by active movements occurring in the protrusions. The suggestion is made that the ground cytoplasm consists of several phases that under normal conditions are miscible to varying extents according to the conditions prevailing. The formation of protrusions with ensuing reversible phase separation is regarded as due to a basic protective mechanism operating in the cytoplasm.

Induction of polyspermy by treatment of sea urchin eggs with mercurials

This study is intended to be a contribution to our knowledge of the mechanism protecting the sea-urchin egg against polyspermy. Various degrees of polyspermy were observed upon insemination of eggs that had been pretreated with the mercurials para-chloro mercuriphenylsulfonic acid (PCMPS) or salicyl-γ-hydroxymercuri-β-methoxypropylamide-0-acetic acid, mersalyl (salyrgan). The exposure of the unfertilized eggs to the reagents lasted usually for 15 min. Thereafter the eggs were washed and inseminated in sea water. The concentration range of the mercurials was approximately 10−7−10−4 M. The effect varied from batch to batch of eggs, indicating a variation of availability of sulfhydryl (SH-) groups in different egg materials. An attempt was made to define a 50 per cent value (−log Km) for the change in the surface that allows one supernumerary spermatozoon to enter. This value would serve the purpose of making experiments with different egg materials more directly comparable (see pp. 162–163). The degree of polyspermy was also dependent on sperm concentration, as exemplified on p. 163. The progress of polyspermy was followed by immersing the eggs for different periods after insemination in 0.001 per cent lauryl sulfate, which stops any further fertilization. Despite the mercurial pretreatment the eggs that remained monospermic developed normally. Pathological development of mercurial-pretreated eggs depended on a polyspermic state (p. 160). Merthiolate had a weaker polyspermy-promoting action than PCMPS and mersalyl. Whereas these latter compounds seemed to act only on the surface layers of the egg, merthiolate (as also p-chloromercuribenzoate and N-ethylmaleimide) also injured the internal processes (p. 167). The effect of mercurials could be removed by a subsequent exposure to cysteine. The concentrations of cysteine necessary were much higher (e.g. 1000 times) than that of mercurial. The mercurials are thus strongly linked, probably to proteins (pp. 167–168). On leaving the eggs in pure sea water after pretreatment with mercurials, their effect is lowered (pp. 168–169). Exposure of the eggs to ~10−4 Mo-iodosobenzoate (IBA) decreased the effect of a following mercurial treatment. This confirms that SH groups are involved in the action of mercurials on the egg surface (pp. 164–165). Exposure to and insemination in the presence of ATP (1.8 × 10−5 M) moderated the effect of pretreatment with mercurial (p. 166). Combination of treatment with IBA prior to the exposure to mercurial and after-treatment with ATP gave a higher moderating effect than that brought about by either IBA or ATP (p. 166). Treatment of the eggs with lower and higher doses of trypsin (see [36]) prior to exposure to mercurials did not modify the extent of polyspermy. The combination of low doses of trypsin and mercurials enhanced the suppression of cleavage beyond the effect of the trypsin doses alone (pp. 169–170). The rate of primary fertilization was not modified by the treatment with mercurials (p. 171). Neither does this treatment modify any microscopic changes accompanying fertilization in such a way that these modifications could be considered as the cause of polyspermy (p. 181). The effect of the mercurials must be at a submicroscopic, biochemical level. On the basis of available knowledge the mechanism of protection against polyspermy and changes in S H compounds upon fertilization are reviewed and discussed (pp. 186–190). The results of mercurial treatment applied soon after fertilization are in keeping with an activation of SH enzymes (pp. 185, 189). The site of attachment of supernumerary spermatozoa and their primary effect give further evidence (see [37]) that, prior to fertilization, a proximaldistal polarity is present in the egg (pp. 177, 189). This polarity also means that the eggs are more receptive to spermatozoa in one region, the proximal one. The cortical changes with ensuing protection evolve also more rapidly in the proximal region [37]. These circumstances are considered to be an important factor in the protection of the egg against polyspermy.

On the effect of adenosine triphosphoric acid and of Ca on the cytoplasm of the egg of the sea urchin, Psammechinus miliaris☆

Abstract The present research aimed at studying the effect of ATP on the cytoplasm of the unfertilized sea urchin egg (Psammechinus miliaris). For this purpose, the eggs were submitted to centrifugation. The time course of stratification was followed in a centrifuge microscope. The curves obtained demonstrate that the rate of stratification is determined rather by rigidity of structure than by viscosity. The addition of 0.003 M ATP increased the rigidity, probably by increasing the linkage between certain components of the cytoplasmic fabric. Upon pretreatment with ATP the structure of the cytoplasm proved to be more coherent after immersion of the eggs in a strongly hypotonic medium. The rigidity proved to be higher in a Ca-free medium than in normal sea water. The rigidity decreased further when the Ca-content of sea water was doubled. Eggs were pretreated with Ca-free sea water and thereafter subjected to strong hypotony. When Ca was added to the hypotonic medium numerous hyaline blisters were given off from the eggs. The blisters arise by a swelling of lipoprotein complexes which in a less swollen state appear as myelin figures. Furthermore the addition of Ca causes a stronger coagulation of the egg surface and of the fibrous framework of the cytoplasm. Addition of ATP causes also blister formation in the eggs cytolyzed in a Ca-free medium. A tendency to coagulation and separation of a membrane recalling the fertilization membrane has been observed upon addition of Ca + ATP. The changes in structure caused by addition of ATP recall those occurring upon the activation of the egg.