Effect of exposure to ribonuclease on the cortical changes occurring upon fertilization: Experiments on sea urchin eggs

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Eggs of Paracentrotus lividus—to a smaller extent also eggs of Psammechinus microtuberculatus—were exposed to solutions of ribonuclease (0.02-0.2-0.36 mg/ml). The eggs were inseminated in the presence of ribonuclease and kept in the enzyme solution for a variable time (in general 30–60 min). The effect of the exposure was examined on living material and and on material that had been fixed in 4 per cent formalin at different lengths of time after fertilization (cf. Table I, for a typical experiment). The RNase treatment brings about a considerable delay in the cortical changes that follow upon fertilization. For more than 2 min the eggs remain in a gelated state with numerous connections between egg surface and elevating fertilization membrane; at the inside of the latter, numerous released (cf. Figs. 6–13) but not incorporated cortical granules were present. In the time interval of 2–10 min gradually one or two concavities developed. Only after concavity formation, did the final incorporation of the released cortical granules in the fertilization membrane take place. Numerous delayed cortical granules were present in the perivitelline space and were here converted into rods or plates. The eggs fertilized in the presence of ribonuclease show a pronounced proximal-distal polarity (Prox.-Dist.). The proximal-distal axis is perpendicular to the animal-vegetal axis (An—Veg.). The Prox.-Dist. axis can be recognized by a concavity or a flattening on the egg surface (cf. Fig. 14 and 15). Moreover, the fertilization membrane is more elevated at the proximal than at the distal pole (this constitutes the main marking of the proximal and the distal region). Also during progress of the first mitosis, the proximal and distal regions can be recognized by a main indentation in the egg cortex (cf. Fig. 22). Later, the polarity in the elevation of the fertilization membrane may be used for this purpose. The site of sperm entry can be recognized in ribonuclease-treated eggs (cf. Fig. 20). There may be a preference for sperm entry at the distal region of the egg (Table II) or the sites of sperm entry may be about equally distributed; in some cases the proximal sperm entry dominated. In control eggs the proximal sperm entry dominated (Table III). The tendency for distal sperm entry is characteristic of eggs exposed to ribonuclease. Variations are due to concentration of enzyme and threshold of suceptibility of the eggs. The sperm entry does not induce the proximal-distal polarity but this is preformed in the egg, which thus has two polar axes that exist prior to fertilization (An.-Veg. and Prox.-Dist.). The rate of fertilization is in general lowered when occurring in the presence of ribonuclease (cf. Figs. 27–29). The birefringent fertilization membrane shows an increase in retardation after exposure to ribonuclease (from about 12.0 to 18.3 mμ). Comparisons were made between the effect of ribonuclease and that of some proteins with a still more basic character (lysozyme and clupein). The difference is so great as to exclude the conclusion that the effect of RNase is mainly due to its basic character. Comparison was made between fully active and only partly active ribonuclease and these experiments point also to the conclusion that the action of RNase is mainly enzymatic. Addition of glutathione shortly before fertilization of ribonuclease-treated eggs counteracts to a large extent the effects of the enzyme (cf. Fig. 34). Of particular interest was the almost complete unfolding of the lamellae of delayed cortical granules after combined treatment with ribonuclease and glutathione (cf. Fig. 35). The experimental results indicate that the exposure to ribonuclease favors release of a factor or factors from ribosomes present in the egg surface; this factor causes an increased gelation of cortical cytoplasm and components of the membranes (fertilization membrane and hyaline layer). The gelation is considered to be due to a polymerization, which in the normal cortical changes may alternate with a depolymerization in a way tentatively indicated in Fig. 41. In this figure the effect of ribonuclease and that of ribonuclease combined with glutathione are also represented. Particularly the changes in the cortical granules, the formation of fertilization membrane and of hyaline layer are discussed in the light of the concept of alternating processes of polymerization and depolymerization. Abnormal conditions induced in different ways are caused by a delay or a break in the transition from one state to the other.