VITELLINE MEMBRANE AND CORTICAL PARTICLES 273
proaches the pH optimum of the cathepsin Β activity. Moreover, a release of basic proteins with subsequent formation of protein complexes
may cause a sensitization of the proteins to cathepsins (Klingenberg and
Walzel, 1951). These enzymes attack proteins at the isoelectric point of
the substrate. By reaction with basic proteins the isoelectric point would
be displaced toward a higher pH value.
The importance of the action of proteolytic enzymes in the cortical
changes is emphasized by the inhibitory effect of ovomucoid and soybean
trypsin inhibitor (Hagström, 1956a). Treatment with these inhibitors
had the effect that a great number of the cortical particles were not
opened and remained in the cortex; no elevation of fertilization membranes occurred in concentrations of 0.003% soybean inhibitor, and the
hyaline layer was poorly developed. The fertilization was Polyspermie.
The spermatozoa caused the formation of a sometimes rather deep hollow
in the egg. The attached spermatozoon was found at the bottom of the
hollow. These hollows are of interest because they show that local displacements of cortical material toward the site of sperm attachment are
mobilized although most of the cortical particles remain unopened.
The nondetached vitelline membrane seems to cover the walls of the
hollows.
Lundblad (1952, Table 9) found in extracts from lyophilized, unfertilized eggs an almost complete inhibition of the enzyme Ε 2 by 0.01%
soybean inhibitor. On the other hand, neither Ε 1 and Ε 3 nor cathepsin
Β were inhibited by this inhibitor (G. Lundblad, personal communication, 1964).
Polyspermy has been noted to occur whenever the opening of the
cortical particles is inhibited as, for example, by pretreatment of the
egg with 2,4-dinitrophenol (Okazaki, 1956), or with butyric acid, lactic
acid, or acetone (Motomura, 1941; Osanai, 1960; Aketa, 1961b).
The results referred to point to the conclusion that the enzyme Ε 2 is a
necessary factor in the opening of the cortical particles. Isolation of the
proteolytic enzymes from the egg (Lundblad and Runnström, 1962;
Lundblad and Lundblad, 1962) will facilitate the testing of the effect of
inhibitors on the proteolytic enzymes contained in the egg.
As mentioned previously, a low dose of trypsin causes a gelation of
the cytoplasm of unfertilized mature eggs of Paracentrotus
(and Psammechinus).
These were exposed, for example, for 15 min to 1 X 10~
4
%
crystalline trypsin; the enzyme was removed by thorough washing, and
the eggs were inseminated. The vitelline membrane was not elevated or
only faintly elevated, but the cortical particles were opened, although
not to the same extent as in the untreated control eggs. The gelated state
of the cytoplasm hindered or delayed the separation of the blastomeres,
proaches the pH optimum of the cathepsin Β activity. Moreover, a release of basic proteins with subsequent formation of protein complexes
may cause a sensitization of the proteins to cathepsins (Klingenberg and
Walzel, 1951). These enzymes attack proteins at the isoelectric point of
the substrate. By reaction with basic proteins the isoelectric point would
be displaced toward a higher pH value.
The importance of the action of proteolytic enzymes in the cortical
changes is emphasized by the inhibitory effect of ovomucoid and soybean
trypsin inhibitor (Hagström, 1956a). Treatment with these inhibitors
had the effect that a great number of the cortical particles were not
opened and remained in the cortex; no elevation of fertilization membranes occurred in concentrations of 0.003% soybean inhibitor, and the
hyaline layer was poorly developed. The fertilization was Polyspermie.
The spermatozoa caused the formation of a sometimes rather deep hollow
in the egg. The attached spermatozoon was found at the bottom of the
hollow. These hollows are of interest because they show that local displacements of cortical material toward the site of sperm attachment are
mobilized although most of the cortical particles remain unopened.
The nondetached vitelline membrane seems to cover the walls of the
hollows.
Lundblad (1952, Table 9) found in extracts from lyophilized, unfertilized eggs an almost complete inhibition of the enzyme Ε 2 by 0.01%
soybean inhibitor. On the other hand, neither Ε 1 and Ε 3 nor cathepsin
Β were inhibited by this inhibitor (G. Lundblad, personal communication, 1964).
Polyspermy has been noted to occur whenever the opening of the
cortical particles is inhibited as, for example, by pretreatment of the
egg with 2,4-dinitrophenol (Okazaki, 1956), or with butyric acid, lactic
acid, or acetone (Motomura, 1941; Osanai, 1960; Aketa, 1961b).
The results referred to point to the conclusion that the enzyme Ε 2 is a
necessary factor in the opening of the cortical particles. Isolation of the
proteolytic enzymes from the egg (Lundblad and Runnström, 1962;
Lundblad and Lundblad, 1962) will facilitate the testing of the effect of
inhibitors on the proteolytic enzymes contained in the egg.
As mentioned previously, a low dose of trypsin causes a gelation of
the cytoplasm of unfertilized mature eggs of Paracentrotus
(and Psammechinus).
These were exposed, for example, for 15 min to 1 X 10~
4
%
crystalline trypsin; the enzyme was removed by thorough washing, and
the eggs were inseminated. The vitelline membrane was not elevated or
only faintly elevated, but the cortical particles were opened, although
not to the same extent as in the untreated control eggs. The gelated state
of the cytoplasm hindered or delayed the separation of the blastomeres,