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

Abstract

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.