VITELLINE MEMBRANE AND CORTICAL PARTICLES 281
and Runnström in 195&-1957, but the data remained unpublished except
for a brief mention by Runnström et al. (1959). In 1957 E. Nakano,
working in conjunction with Runnström (unpublished results), found in
studies on Paracentrotus
that a noninhibitory concentration of chondroitin sulfate (0.05-0.1%) became inhibitory after eliminating the
vitelline membrane by exposure to trypsin or chymotrypsin. Dextran
sulfate (that even in 0.5% had no, or a low, inhibitory effect on intact
eggs) proved inhibitory to trypsin-pretreated eggs. Sugiyama (1951)
showed that fertilized eggs may be refertilized if the fertilizaton membrane and the hyaline layer are removed after the first fertilization. It
was demonstrated in Nakano's work that the re fertilization process is
still more sensitive to the action of the sulfated mucopolysaccharides
than the eggs pretreated with trypsin. Exposure to 0.1% chondroitin sulfate allowed only 33% refertilization, whereas in pure sea water 77% of
the eggs were refertilized. When intact eggs were used, 100% fertilization
was obtained both in control and in chondroitin sulfate tests. It is thus
evident that the sulfated mucopolysaccharides react with the cytoplasmic
surface. The bonds responsible for the inhibitory action must be very
loose, because the inhibition can be removed simply by washing the
treated eggs with sea water.
Nakano (1956) dissolved the hyaline layer in molar urea, spun down
the eggs, and precipitated the substance of the hyaline layer with ammonium sulfate. It proved to contain mucopolysaccharide and protein
with an admixture of lipid. Brought into sea water solution it exerted an
inhibitory action on refertilization of eggs of Hemicentrotus
lividus.
Previous autoclaving of the substance of the hyaline layer increased the
inhibitory action. This agrees completely with the increase in inhibitory
activity that Harding (1951) found after autoclaving a rather weakly
acting heparin preparation. Moreover, Nakano (loc. cit.) stated that
addition of 5 Χ ΙΟ
-3 M NaI0 4 completely removed the inhibitory action
of the dissolved substance of the hyaline layer. This is again in harmony
with the results of Runnström and Kriszat (1950b) and Harding (1951)
referred to above, in which NaI0 4 removed the inhibitory action of jelly
coat substance and heparin.
In his work with Paracentrotus,
Nakano demonstrated that the substance of the hyaline layer had no effect on the fertilization of intact
eggs, but that inhibition is pronounced on eggs pretreated with trypsin,
or fertilized eggs deprived of fertilization membrane and hyaline layer
(refertilization). Also the substance of the hyaline layer preferably
acts, thus, directly on the cytoplasmic egg surface.
Nakano included in the investigation on Paracentrotus
also some experiments in which porphyrexid was used as an inhibitor. Here he found
and Runnström in 195&-1957, but the data remained unpublished except
for a brief mention by Runnström et al. (1959). In 1957 E. Nakano,
working in conjunction with Runnström (unpublished results), found in
studies on Paracentrotus
that a noninhibitory concentration of chondroitin sulfate (0.05-0.1%) became inhibitory after eliminating the
vitelline membrane by exposure to trypsin or chymotrypsin. Dextran
sulfate (that even in 0.5% had no, or a low, inhibitory effect on intact
eggs) proved inhibitory to trypsin-pretreated eggs. Sugiyama (1951)
showed that fertilized eggs may be refertilized if the fertilizaton membrane and the hyaline layer are removed after the first fertilization. It
was demonstrated in Nakano's work that the re fertilization process is
still more sensitive to the action of the sulfated mucopolysaccharides
than the eggs pretreated with trypsin. Exposure to 0.1% chondroitin sulfate allowed only 33% refertilization, whereas in pure sea water 77% of
the eggs were refertilized. When intact eggs were used, 100% fertilization
was obtained both in control and in chondroitin sulfate tests. It is thus
evident that the sulfated mucopolysaccharides react with the cytoplasmic
surface. The bonds responsible for the inhibitory action must be very
loose, because the inhibition can be removed simply by washing the
treated eggs with sea water.
Nakano (1956) dissolved the hyaline layer in molar urea, spun down
the eggs, and precipitated the substance of the hyaline layer with ammonium sulfate. It proved to contain mucopolysaccharide and protein
with an admixture of lipid. Brought into sea water solution it exerted an
inhibitory action on refertilization of eggs of Hemicentrotus
lividus.
Previous autoclaving of the substance of the hyaline layer increased the
inhibitory action. This agrees completely with the increase in inhibitory
activity that Harding (1951) found after autoclaving a rather weakly
acting heparin preparation. Moreover, Nakano (loc. cit.) stated that
addition of 5 Χ ΙΟ
-3 M NaI0 4 completely removed the inhibitory action
of the dissolved substance of the hyaline layer. This is again in harmony
with the results of Runnström and Kriszat (1950b) and Harding (1951)
referred to above, in which NaI0 4 removed the inhibitory action of jelly
coat substance and heparin.
In his work with Paracentrotus,
Nakano demonstrated that the substance of the hyaline layer had no effect on the fertilization of intact
eggs, but that inhibition is pronounced on eggs pretreated with trypsin,
or fertilized eggs deprived of fertilization membrane and hyaline layer
(refertilization). Also the substance of the hyaline layer preferably
acts, thus, directly on the cytoplasmic egg surface.
Nakano included in the investigation on Paracentrotus
also some experiments in which porphyrexid was used as an inhibitor. Here he found