260
JOHN RUNNSTRÖM
dealt with by J. C. Dan (1952). Dan (1954a,b) considered that the
acrosomal filament attaches the spermatozoon to the vitelline membrane of the egg. This is corroborated by electron micrographs of Afzelius
and Murray (1957). Before reaching the plasma membrane, the spermatozoon must penetrate the vitelline membrane. When the eggs were fixed
in 4% formaldehyde some 10 sec after insemination, the vitelline membrane was elevated in a small region around the attached spermatozoon
which is lifted up along with the membrane. At somewhat later fixation
(15-20 sec after insemination) the vitelline membrane alone was elevated, whereas the spermatozoon had attached itself to the cytoplasmic
surface. In the living sea urchin material (Paracentrotus
lividus) a certain local delamination of the vitelline membrane around the attached
spermatozoon may occur probably within 10 sec after insemination at
room temperature (20°C). Fixation in formaldehyde causes a certain
shrinkage that brings about the elevation of the vitelline membrane
along with the spermatozoon. This elevation is thus a fixation artifact,
which, however, shows that a break in the intimate connection between
vitelline membrane and plasma membrane has already occurred
(Runnström et al, 1959). It may be remarked in passing that the timing
of the different steps in the cortical changes refers to gametes that have
been mixed efficiently so that the time for attainment of egg-sperm
contact may be considered to be negligible.
The method of Hagström and Hagström (1954) allows evaluation
of the rate of effective attachment of the spermatozoa to the egg surface
after insemination. Hagström (1959) showed that pretreatment of the
unfertilized eggs of several urchin species with 0.001% trypsin for 10
min resulted in a considerable decrease in the rate of fertilization. In one
experiment with Psammechinus
microtuberculatus
the 50% fertilization
time was prolonged from 20 sec in the control to 80 sec in eggs pretreated
for 10 min with 1 X 10~
3
% crystalline trypsin. In experiments reported
by Hagström (loc. cit.) on gametes of Paracentrotus
lividus the 50%
fertilization time was still more prolonged. The effect of trypsin on the
fertilization rate varies in different batches of eggs. Runnström and
Kriszat (unpublished data, 1964) found an effect on the fertilization
rate even after pretreatment of the eggs for 15 min with 1 X 10
_ 4
%
twice crystallized trypsin (ca. 12 BAEE units per milliliter). In several
experiments the 50% fertilization time was prolonged about twofold.
Runnström and Kriszat (1960) found that after pretreatment with
higher concentration of trypsin, e.g. 3-6 X 10
_ 4
% trypsin, a certain number of eggs remained unfertilized. It was demonstrated that the nonfertilizable eggs could still be activated by exposure for 5 min to ΙΟ
-4 M
sodium periodate in sea water. These results pointed to the presence of
JOHN RUNNSTRÖM
dealt with by J. C. Dan (1952). Dan (1954a,b) considered that the
acrosomal filament attaches the spermatozoon to the vitelline membrane of the egg. This is corroborated by electron micrographs of Afzelius
and Murray (1957). Before reaching the plasma membrane, the spermatozoon must penetrate the vitelline membrane. When the eggs were fixed
in 4% formaldehyde some 10 sec after insemination, the vitelline membrane was elevated in a small region around the attached spermatozoon
which is lifted up along with the membrane. At somewhat later fixation
(15-20 sec after insemination) the vitelline membrane alone was elevated, whereas the spermatozoon had attached itself to the cytoplasmic
surface. In the living sea urchin material (Paracentrotus
lividus) a certain local delamination of the vitelline membrane around the attached
spermatozoon may occur probably within 10 sec after insemination at
room temperature (20°C). Fixation in formaldehyde causes a certain
shrinkage that brings about the elevation of the vitelline membrane
along with the spermatozoon. This elevation is thus a fixation artifact,
which, however, shows that a break in the intimate connection between
vitelline membrane and plasma membrane has already occurred
(Runnström et al, 1959). It may be remarked in passing that the timing
of the different steps in the cortical changes refers to gametes that have
been mixed efficiently so that the time for attainment of egg-sperm
contact may be considered to be negligible.
The method of Hagström and Hagström (1954) allows evaluation
of the rate of effective attachment of the spermatozoa to the egg surface
after insemination. Hagström (1959) showed that pretreatment of the
unfertilized eggs of several urchin species with 0.001% trypsin for 10
min resulted in a considerable decrease in the rate of fertilization. In one
experiment with Psammechinus
microtuberculatus
the 50% fertilization
time was prolonged from 20 sec in the control to 80 sec in eggs pretreated
for 10 min with 1 X 10~
3
% crystalline trypsin. In experiments reported
by Hagström (loc. cit.) on gametes of Paracentrotus
lividus the 50%
fertilization time was still more prolonged. The effect of trypsin on the
fertilization rate varies in different batches of eggs. Runnström and
Kriszat (unpublished data, 1964) found an effect on the fertilization
rate even after pretreatment of the eggs for 15 min with 1 X 10
_ 4
%
twice crystallized trypsin (ca. 12 BAEE units per milliliter). In several
experiments the 50% fertilization time was prolonged about twofold.
Runnström and Kriszat (1960) found that after pretreatment with
higher concentration of trypsin, e.g. 3-6 X 10
_ 4
% trypsin, a certain number of eggs remained unfertilized. It was demonstrated that the nonfertilizable eggs could still be activated by exposure for 5 min to ΙΟ
-4 M
sodium periodate in sea water. These results pointed to the presence of