Georg Kriszat

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.

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.

The activation of the egg of the sea urchin Psammechinus miliaris by periodate

Abstract Sodium periodate in concentration 6.10−5–9.10−5 N has an activating effect on the eggs of Psammechinus miliaris. A membrane elevation occurred and cleavage started. It did not proceed beyond the 2–8 cell stage. The eggs activated subsequent to the periodate treatment exhibited the angular plasmolysis characteristic of fertilized eggs when immersed in a hypertonic medium. Even non activated periodate treated eggs behave differently from non pretreated eggs. The latter are often somewhat elongated in one direction and show a fine wrinkling on the surface. The former are spherical with a smooth surface. This behaviour indicates that the eggs have taken a step towards activation. Eggs activated subsequent to periodate treatment are as strongly stained with methylene blue as are fertilized eggs. In contrast the non activated pretreated or the non pretreated unfertilized eggs are very weakly or not at all stained with methylene blue. Iodate of corresponding concentrations has a much lower activating effect than sodium periodate. The activation of the eggs by periodate occurred even in absence of Ca. After transfer to Ca-containing medium the eggs underwent a contraction and the membrane became much more refractile.

The gelating effect of lower doses of trypsin on sea urchin eggs and its removal by exposure to glutathione

Eggs of Paracentrotus lividus or Psammechinus miliaris were exposed before fertilization to different doses of trypsin with results similar to those reported earlier (cf. [12]). With lower doses of trypsin a gelation was brought about which blocked the cleavage of the pretreated eggs. If, however, the trypsin pretreated unfertilized eggs were subsequently exposed to 2 × 10−3 M glutathione (e.g. for 30 min) the trypsin effect manifested after fertilization was lowered or removed. Histidine of the same molar concentration had almost no improving effect. The results indicate that the trypsin pretreatment promotes the activation of proteolytic enzymes present in the eggs. These are of two types. The first one has a gelating or polymerizing action on certain cytoplasmic components; it is activated by pretreatment with low doses of trypsin. Glutathione has, however, an activating effect on another system of proteolytic enzymes that has a more pronounced hydrolytic action; in this way, a depolymerization is brought about with subsequent lowering of the degree of gelation in the cytoplasm. Glutathione may also exert an inhibitory effect on the gelating enzyme system. The action of glutathione is similar to that brought about by an increase in trypsin (cf. Tables I and II). Changes in the balance between the two enzyme systems mentioned may play a role in cellular events. Certain additional observations were recorded concerning the strong phase separation occurring in nondividing fertilized eggs, pretreated with low doses of trypsin [12]. In other eggs a milder separation occurs that may be compatible with cleavage. These eggs often form a uniform bulge which penetrates through a hole in an adhering fertilization membrane. The eggs with a bulge have a sticky surface; this circumstance leads to the formation of aggregates of both eggs and spermatozoa. Unfertilized eggs do not undergo phase separation when kept in sea water after trypsin treatment. A phase separation is, however, induced in unfertilized eggs if they come in intimate contact with fertilized eggs that have undergone a phase separation compatible with segmentation. This must mean that a transfer of an agent occurs which induces phase separation. This may involve a step toward activation of the unfertilized egg as certain reorientations in the cytoplasm indicate. The effects on eggs exposed to low doses of trypsin and on those exposed to ribonuclease [14] were compared. A brief survey was also made of the consequences of exposure to these enzymes to ensuing development. In both cases the enzymes provoke the activation of enzyme(s) causing an increase in consistency, a gelation, in the cytoplasm. Different phases are, however, involved in the response. This is regarded as the cause of the rather important differences that are observed when the effects of the two enzymes on cleavage and development are compared.

The solidifying effect of certain oxidation reduction indicators on the surface of the unfertilized sea urchin egg.

Abstract Further studies have been carried out on the effect of porphyrindin on the egg of the sea urchin, Psammechinus miliaris . To a smaller extent also eggs of Paracentrotus lividus were studied. Eggs suspended on the border between sea water and isotonic sucrose solution were centrifuged under certain standard conditions. Following treatment with porphyrindin the eggs elongated very slightly and an equilibrium state was soon attained. The elongation was stronger in the control eggs and the equilibrium was attained only after a longer interval of time. Conversely, the stratification in the interior was alike in control eggs and those subjected to porphyrindin. The results indicate that the rigidity of the egg surface but not that of the interior of the egg is increased subsequent to the treatment with porphyrindin. Observations on the plasmolysis of eggs in a hypertonic solution also pointed to an increased rigidity of the surface in eggs subjected to porphyrindin as compared with control eggs. Pretreatment of unfertilized eggs with crystalline trypsin removed to a considerable extent the effect of a temporary exposure to porphyrindin. If such eggs were fertilized a much higher percentage developed into blastulae of normal type than when the eggs had been subjected to porphyrindin without pretreatment with trypsin. Both trypsin and porphyrindin attack in an irreversible way metaplasmatic structures in the surface of the unfertilized egg, particularly the vitelline membrane. If this membrane has been removed by trypsin treatment the direct effect of porphyrindin is on the cytoplasmatic cortex but this effect is reversible at least to a certain extent depending on concentration of porphyrindin and duration of the exposure. Even a temporary exposure of trypsin-pretreated eggs to porphyrindin was sometimes observed to impair the propagation of the impulse of activation. Partially developed eggs and blastulae then resulted. The possibility is considered that the oxidation-reduction state is involved in the propagation of the impulse. When insemination was carried out in the presence of porphyrindin, fertilization was inhibited also in eggs pretreated with trypsin. In certain experiments unfertilized eggs were first pretreated with porphyrindin and only afterwards subjected to trypsin. The treatment with porphyrindin protects the egg surface against the action of trypsin. Under these conditions the vitelline membrane may resist the trypsin treatment whereas it is broken up by trypsin in non-porphyrindin-pretreated eggs. Sometimes the trypsin treatment deprives the egg of the capacity of being fertilized. Porphyrindin may protect against this effect of trypsin.