liation
chem-
27
100
aot
i
60b
7
40+
4
43 30+
4
Oo
<I
>
a
55
az
vz
az 20
=
4
—
>
za
=a
<
a
<
1g} —
J
6t
L
4
0
_
10
l
20
l
30
l
40
RECOVERY PERIOD IN MINUTES
l
50
60
Fic. 22.—Decrease in cleavage delay in Arbacia sperm ex| posed to 7200R as a function of the time the control cell
Fe division cyele is prolonged by treatment of the zygote with
. cither beta mercaptoethanol or EDTA.
sugof the
takes
Id be ) Ocean{) continuously through a calcareous filtrant
f the | spread over the bottom of the tank.
o the
Gametes are procured by injection of 0.6 M KCI or
t peby electrical stimulation of the animals with an aleggs
ternating potential of about 8 volts. The fertilized
; and - eggs develop in either Instant Ocean or natural sea
. and
water at room temperature. The radiation doses are
| dif- ' delivered by a #87Cs gamma source at a dose rate of
also
about 1200 R/min.
1 the
Since the first division of fertilized sea urchin eggs
is so remarkably synchronous, estimation of the time
This
when 50% of the eggs have cleaved is made visually
disunder low magnification. This time can generally be
the
dermined to within +1 min. Hence, cleavage delays
ly of
are good to about +2 min.
s of
sible
RESULTS
cell
acia
nals
aria
sant
yods
‘ida.
vali-
difference in time of action. These agents are listed in
modes of action. That recovery has been observed
__|
8}
fertilized eggs at 0° C or any temperature lower than
normal. Two other agents, EDTA and beta mercaptoethanol, have now been used which prolong the cycle
and also permit recovery to take place. Figure 22
shows the effect of treating the same gametes with
these agents. At the concentrations used, EDTA appears to be more effective, probably because of less
interference with activity required for recovery or a
Table 6 along with the others and their assumed
0.1 M BETAMERCAPTOETHANOL
Oo
tion-induced division delay is evident, however, when
the cell eycle time is extended by incubation of the
with all these agents except temperature must indicate that the temperature coefficient of the recovery
process is similar to that applying to extension of the
cell cycle, Additional evidence is provided by the ob-
servation that the magnitude of the radiation-induced
division delay in any one experiment is independent
of the temperature (between 8° C and room temperature) at which the fertilized eggs develop.“
In order to define the time of action of the agents
listed in Table 6, their effects on the radiation sensitivity curve (Figure 21) were determined. The
TABLE 6
Errect oF Various AGENTS ON CELL
DIvIsion CYCLE
Agent inducing
prolongation of
division cycle
Effect on
radiationinduced
cleavage
delay
0 to 15.5° C
D'~D
N,
D' < D{
D,0
D' < D
Puromycin (+N:2)
D’ < D
NaN3
D’ < D
Colcemid
D’ < D
Ribonuclease
D’ < D
°
D’ < D
Previous publications’*® have described the ef-
fects of prolonging the control cell division cycle by
temporary exposure of fertilized eggs to nitrogen,
D.0O, sodium azide, Coleemid, ribonuclease, or 0° C on
the radiation-induced cleavage delay. When the cell
cycle is prolonged by treatment with all these agents
except low temperature, there is a decrease in the
radiation-induced cleavage delay. Recovery is, there-
fore, said to have occurred during the time the cyele
Is
prolonged. No significant reduction in the radiaeh
+ Supplied by Aquarium Systems,Inc., Wickliffe, Ohio.
EDTA
Interpretation
No recovery when division
period prolonged by reduced
temperature.
Aerobic metabolism not re-
quired for recovery.
Rigidification of mitotie apparatus does not interfere
with recovery.
Recovery not wholly dependent on protein synthesis.
Aerobic metabolism not re-
quired for recovery.
Interference with spindle formation does not interfere
with recovery process.
Surface effect? Does not interfere with recovery.
Complexing of divalent cations?
Beta mercaptoethanol
D’ < D
Does not
interfere
with recovery.
Interference with aster and
spindle development does
not block recovery process.
D = Radiation-induced cleavage delay.
D’ = Radiation-induced cleavage delay after treatment
with agent inducing recoveryperiod.